chromatography_bioprocessiii.pdf
TRANSCRIPT
Bioprocess-III (Detailed Chromatography)
Prepared by Mohd Tayyab and Nimish Gopal IIT Roorkee
(AEC)
Anion exchange chromatography
Anion exchange chromatography bull Beads are +vely charged
bull Eg DEAE-C (Diethylaminoethyl cellulose) matirx
bull -vely charged proteins bind to beads
bull +vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for acidic proteins
bull Acidic proteins have low pI
bull Why for acidic If you want to use it for basic hellipvery high pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
(Sulfate derivative) Carboxymethyl (CM)
Quaternary amine Diethylaminoethyl (DEAE)
Anion exchange chromatography beads (matrixresins)
IEC Groups
Strong Anion Exchanger Charged (ionized) across a
wide range of pH levels Permanantly charged
Weak Anion Exchanger Charged within a
narrower pH range Charge dependent on pH
Above 90 not good
Cellulose Matrix Exchange Type Aminoethyl-Cellulose Weak
Diethylaminoethyl (DEAE)-Cellulose Weak
Benzyl DEAE-Cellulose Weak
Polyethylenimine (PEI)-Cellulose Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
Strong
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Commonly Used Anion-Exchange Matrices for Protein Seperation
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
(AEC)
Anion exchange chromatography
Anion exchange chromatography bull Beads are +vely charged
bull Eg DEAE-C (Diethylaminoethyl cellulose) matirx
bull -vely charged proteins bind to beads
bull +vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for acidic proteins
bull Acidic proteins have low pI
bull Why for acidic If you want to use it for basic hellipvery high pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
(Sulfate derivative) Carboxymethyl (CM)
Quaternary amine Diethylaminoethyl (DEAE)
Anion exchange chromatography beads (matrixresins)
IEC Groups
Strong Anion Exchanger Charged (ionized) across a
wide range of pH levels Permanantly charged
Weak Anion Exchanger Charged within a
narrower pH range Charge dependent on pH
Above 90 not good
Cellulose Matrix Exchange Type Aminoethyl-Cellulose Weak
Diethylaminoethyl (DEAE)-Cellulose Weak
Benzyl DEAE-Cellulose Weak
Polyethylenimine (PEI)-Cellulose Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
Strong
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Commonly Used Anion-Exchange Matrices for Protein Seperation
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Anion exchange chromatography bull Beads are +vely charged
bull Eg DEAE-C (Diethylaminoethyl cellulose) matirx
bull -vely charged proteins bind to beads
bull +vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for acidic proteins
bull Acidic proteins have low pI
bull Why for acidic If you want to use it for basic hellipvery high pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
(Sulfate derivative) Carboxymethyl (CM)
Quaternary amine Diethylaminoethyl (DEAE)
Anion exchange chromatography beads (matrixresins)
IEC Groups
Strong Anion Exchanger Charged (ionized) across a
wide range of pH levels Permanantly charged
Weak Anion Exchanger Charged within a
narrower pH range Charge dependent on pH
Above 90 not good
Cellulose Matrix Exchange Type Aminoethyl-Cellulose Weak
Diethylaminoethyl (DEAE)-Cellulose Weak
Benzyl DEAE-Cellulose Weak
Polyethylenimine (PEI)-Cellulose Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
Strong
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Commonly Used Anion-Exchange Matrices for Protein Seperation
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
(Sulfate derivative) Carboxymethyl (CM)
Quaternary amine Diethylaminoethyl (DEAE)
Anion exchange chromatography beads (matrixresins)
IEC Groups
Strong Anion Exchanger Charged (ionized) across a
wide range of pH levels Permanantly charged
Weak Anion Exchanger Charged within a
narrower pH range Charge dependent on pH
Above 90 not good
Cellulose Matrix Exchange Type Aminoethyl-Cellulose Weak
Diethylaminoethyl (DEAE)-Cellulose Weak
Benzyl DEAE-Cellulose Weak
Polyethylenimine (PEI)-Cellulose Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
Strong
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Commonly Used Anion-Exchange Matrices for Protein Seperation
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
(Sulfate derivative) Carboxymethyl (CM)
Quaternary amine Diethylaminoethyl (DEAE)
Anion exchange chromatography beads (matrixresins)
IEC Groups
Strong Anion Exchanger Charged (ionized) across a
wide range of pH levels Permanantly charged
Weak Anion Exchanger Charged within a
narrower pH range Charge dependent on pH
Above 90 not good
Cellulose Matrix Exchange Type Aminoethyl-Cellulose Weak
Diethylaminoethyl (DEAE)-Cellulose Weak
Benzyl DEAE-Cellulose Weak
Polyethylenimine (PEI)-Cellulose Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
Strong
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Commonly Used Anion-Exchange Matrices for Protein Seperation
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Cellulose Matrix Exchange Type Aminoethyl-Cellulose Weak
Diethylaminoethyl (DEAE)-Cellulose Weak
Benzyl DEAE-Cellulose Weak
Polyethylenimine (PEI)-Cellulose Weak
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
Strong
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak
Commonly Used Anion-Exchange Matrices for Protein Seperation
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Polystyrene Matrix Exchange Type Analytical Grade (AG)-1 Strong Analytical Grade (AG)-2 Strong Bio-Rex 5 Intermediate AG 3-X4A Strong Bio-Rex MSZ 1-X8 Strong
Sephadex Matrix Exchange Type Diethylaminoethyl (DEAE)-Sephadex Weak Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
Strong
Sepharose Matrix Exchange Type Diethylaminoethyl (DEAE)-Sepharose CL-6B Weak Diethylaminoethyl (DEAE)-Sepharose (fast flow) Weak Quaternary amine (Q)-Sepharose (fast flow) Strong
Commonly Used Anion-Exchange Matrices for Protein Seperation
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Factors considered for choosing IEC matrix (1) pH Range Where the Protein of Interest is Stable If the protein is most stable at pHs above its pI then an anion-exchange gel matrix should be used If it is most stable below its pI then a cation-exchange gel matrix should be used (2) Molecular Size of the Protein being Separated The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix For proteins of molecular weight 10000 to 100000 DEAE-Sephacel and DEAE-Sepharose are good choices For larger proteins Sephadex A-25 or C-25 which have the highest charge density at the surface of the gel are appropriate (3) Operating Pressue For nonrigid gels (eg Sephacel or Sepharose) operating pressures should not exceed the manufacturer defined limits For rigid gels (eg Analytical grade (AG) Bio-Rex MSZ and Dowex commercial grade ion-exchange resins from Bio-Rad) packing can be carried out at higher flow rates using a peristaltic pump (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix The column diameters most frequently used are 1 2 and 25 cm Reservoir design direction of column flow and whether or not a peristaltic pump should be employed are arbitrary Separating columns are usually lt20 cm in length If the protein mixture is exceedingly complex a longer column should be used
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Cl- is exchanged with protein
Mobile Counter Ions in Anion exchange
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Steps of Anion Exchange chromatography (AEC)
I Set up column other equipment
II Choose buffer (pH important above protein pI for AEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Chromatogram
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
What MakesProteins Adsorb And Desorb From The Column
bull Proteins have charges because their amino acids are charged
bull At a given pH different proteins have different charges because they are composed of different amino acids
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Charge On Proteins
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Some amino acids have R group with positive charge others negative charge others no charge
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
In theory IEC can separate proteins that differ by only one charged group Or Two protein both negatively charged will bind anion exchange matrix Can both be separated
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
pI Is Important
bull First pI for a protein of interest determines whether use cation or an anion resin
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 62 may want to use a buffer at 72
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
IEC Principles
Order of elution +vely will not bind and negatively charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Chloride ions (NaCl) bullIncreasing concentration bullFirst least negatively charged proteins will elute bullFollowed by more negaitvely charged proteins OR With pH gradient bullLow the pH slowly bullProteins will slowly become less negative and than positive as pH will reach below its pI
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
(CEC)
Cation exchange chromatography
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Cation exchange chromatography (CEC) bull Beads are -vely charged
bull Eg Carboxymethyl (CM) matrix
bull +vely charged proteins bind to beads
bull -vely charged proteins will elute out
bull Add buffer to elute unbound proteins
bull Bound protein are eluted by changing pH or by passing a gradually increasing linear salt gradient (NaCl high concentration)
bull Generally used for basic proteins
bull pH of mobile phasebuffer used is 4 - 7
bull Basic proteins have high pI
bull Why for basic If you want to use it for acidic hellipvery low pH may denature proteinhelliplose its activity
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Matrix polymeric porous beads bullCellulose bullSepharose is a tradename for a crosslinked beaded-form of a polysaccharide polymer material extracted from seaweed It is crosslinked through lysine side chains bullSephacel bead form of cellulose bullSephadex crosslinked dextranbead form of dextran
Packed (poured) into the column as a slurry
Matrix Is Support Covalently attached functional group (charged)
Matrix or Resin
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
CEC Groups Cation exchange chromatography beads (matrixresins)
Strong Cation Exchanger Charged (ionized) across a
wide range of pH levels Permanently charged
Weak Cation Exchanger Charged within a
narrower pH range Charge dependent on pH
below 40 not good
(Sulfate derivative) Carboxymethyl (CM)
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
CEC Groups
ƒ Cation ECnegative matrix
CM = carboxymethyl cellolusesephadex SP = sulphopropyl S = sulphonate
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Cellulose Matrix Exchange Type Carboxymethyl (CM)-Cellulose Weak
Polystyrene Matrix Exchange Type AG 50W (sulphonated) Strong Bio-Rex 70 (carboxylic acid) Weak
Sephadex Matrix Exchange Type Carboxymethyl (CM)-Sephadex Weak Sulphopropyl (SP)-Sephadex Strong
Sepharose Matrix Exchange Type Carboxymethyl (CM)-Sepharose CL-6B Weak Carboxymethyl (CM)-Sepharose (fast flow) Weak Sulphonate (S)-Sepharose (fast flow) Strong
Commonly Used cation-Exchange Matrices for Protein Separation
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
bullIons that move bullAdded in buffer solution bullFlow through the column bullInteract with charged groups on the beads bullIncreasing concentration of NaCl added bull Na+ is exchanged with protein
Mobile Counter Ions in cation exchange
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Steps of Cation Exchange chromatography (CEC)
I Set up column other equipment
II Choose buffer (pH important below protein pI for CEC)
III Prepare resin pour column equilibrate resin with buffer
IVLoad sample onto column --adsorption
V Wash unbound proteins off the column
VI UV spectrophotometer= OD 280 nm
VIIDesorption elution (step gradientcontinous gradient) (Collect fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIIIEnd desorption regenerate column (optional)
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Analysis of elution profile (chromatogram)
Analyze sample for purity on SDS-PAGE
Pool fractions containing pure samples
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Chromatogram Elution profile showing absorbance at 280 on y axis and
fraction number on x axis
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Charge On Proteins that bind CEC
bull Overall net charge on a protein is determined by the R groups of its amino acids
ndash Basic protein + amino acids have R group with more positive charge and less negative charge
ndash pI is generally above 7
ndash pH range used for CEC 4-7
ndash Protein will be +vely charged
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
In theory IEC can separate proteins that differ by only one charged group Or Two protein both +vely charged will bind cation exchange matrix Can both be separated Yes With different salt (NaCl) conc in elution buffer How
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
pH Of Environment Is Crucial
bull pH affects stability of proteins
bull pH of environment affects charge on a protein
bull pH is controlled with the buffer chosen
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
pI
bull Also usually donrsquot want to use a pH where the protein has no charge at all ndash because it will never stick to the beads
bull So for example pI is 80 may want to use a buffer at 68 for cation exchange
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
IEC Principles Cation exchange
Order of elution -vely will not bind and +vely charged protein will bind
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
IEC Principles
Mehods of Elution desorption of proteins bound to anion exchange With Salt Gradient bullCounter anion Sodium ions (NaCl) bullIncreasing concentration bullFirst least +vely charged proteins will elute bullFollowed by more +vely charged proteins OR With pH gradient bullIncrease the pH of buffer slowly bullProteins will slowly become less positive and than -ve as pH will reach above its pI and elute
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Protein purification system (AKTA purifier) FPLC (fast protein liquid chromatography)
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
CEC chromatogram (pH increase leading to elution)
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity Chromatography bull Adsorptive separation in which the molecule
to be purified specifically and reversibly binds (adsorbs) to a specific chemical group (a ligandprosthetic groupsubstrateproductanalog of substarteproduct) immobilized on an insoluble support (a matrix or resin)
bull Purification is 1000X or better from a single step (highest of all methods)
bull Preferred method if possible
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the proteins biological activity These proteins are commonly enzymes and cofactors can be considered helper molecules that assist in biochemical transformations Eg Metal ions Vitamins Hemoglobin (Heme + Fe)
Ligandprosthetic group
bull Cofactors are either organic or inorganic bull Loosely-bound cofactors termed coenzymes bullTightly-bound cofactors termed prosthetic groups
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity Chromatography
Step 1 Attach ligand X to column matrix
Step 2 Load protein mixture onto column
X
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity Chromatography
Step 3 A specific protein binds to the ligand
Step 4 Wash column to remove unwanted Material Step 5 Elute bound protein later by providing ligand in free form in buffer (or by changing pH or Salt concentration) Change in pH and salt concentration breaks reversible interaction between protein and ligand
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity Chromatography
bull Used in many applications
bull Purification of substances from complex biological mixtures
bull Separation of native from denatured forms of proteins (why)
bull Removal of small amounts of biomaterial from large amounts of contaminants
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity Chromatography
bull The ligand must be readily (and cheaply) available
bull Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein
bull Binding must not be too strong or weak
bull Elution involves passage of high salt or low pH buffer after binding or unbound ligand in buffer
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Example of affinity puriifcation
bull Lectin proteins bind specifically to sugar molecules
bull Present in seeds of various plants
bull Ligand Glucose
bull Protein eluted by addition of concentrated solution of glucose
bull Glucose in solution displaces column attached glucose molecule from binding site in protein
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity chromatography
bull Enzyme-substrate binding
bull Antibody-antigen binding
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Immunoaffinity Chromatography A type of Affinity chromatography bullUses antibodies bullAntibodies bind very specifically to antigens bullAntigen is injected in organism (Horserabbitmouse) bullAntibodies are made and present in blood bullAb are purified from blood bullPurified Ab are conjugated to beads bullSample containing the antigen is passed through the affinity column bullAntigen will bind to antibodies conjugated to beads bullElution of bound protein is done by lowering the pH to 30
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Immunoaffinity Chromatography Antigen and Antibody interaction
Complementary shape and reversible interaction
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Immunoaffinity Chromatography Uses Immunoaffinity columns (IACs) Small molecules can also be purified
Disadvantage column can not be reused antibody is denatured due to low pH
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Affinity Chromatography using Protein Affinity Tags
Protein tags are peptide sequencesprotein genetically fused with a recombinant protein Often these tags are removable by chemical agents or by protease enzymatic means Affinity tags are attached to proteins so that they can be easily purified in one step using affinity chromatography technique Most common affinity tags bull Poly-His tag bull Glutathione-S-transferase (GST) bull Maltose binding protein (MBP) bull Chitin binding protein (CBP)
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Protein Affinity Tags
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Immobilized metal ion affinity chromatography (IMAC) Ni affinity chromatography
Chelator
Allows proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions such as cobalt nickel copper for the purification of histidine tag containing proteins or
peptides
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Elution of Histag protein bound to Ni column
1 By decreasing the pH 2 By running linear gradient of imidazole
Imidazole Histidine
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Steps of Ni ion affinity chromatography
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Glutathione S-transferase (GST) tags
bullGST is a 35-KDa protein bullGST has an affinity for glutathione bullAvailable immobilized as glutathione agarose bullAn excess amount of gluthione is used to displace the tagged protein for elution bullThe glutathione resins selectively bind to GST-tagged proteins effectively allowing the specific protien of interest be separated from the mixture at high efficiency
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Step of GST-tagged protein purification by affinity chromatography
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
MBP-Tag bullMaltose binding protein (MBP) tag bullMaltose in buffer bullDisaccharide of two glucose
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin
kallikrein clostripain
Cibacron Blue
Dye
Serum Albumin Preablumin
Heparin Growth factors cytokines coagulation
factors
Protein A Fc region of immunoglobulins
Calmodulin Calmodulin regulated kinases cylcases
and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Size Exclusion Chromatography
bull Molecules are separated according to differences in their size as they pass through the gel-filtration matrix
bull Non-adsorption technique
bull Ideally no interaction between matrix and molecules
bull Polymer beads composed of cross-linked dextran (dextrose) which is highly porous
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
SEC bull Molecules with diameter greater than the largest
pores within resin material are unable to enter the particle Therefore they pass through the smallest accessible volume they travel through the column from top to bottom fastest and elute first
bull Smaller molecules enter pores and access pores within the resin particles Pass through the larger accessible volume within the column and beads Therefore elute later
bull Elution is in order of decreasing molecular weight
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Size Exclusion Chromatography (SEC)
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
SEC
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Sephadex Structure Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex branched glucan (polysaccharide made of many glucose molecules)
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
SEC Gel-filtration chromatography Determination of protein molecular weight
Load and run molecular weight markers Draw standard curve
Load and run protein sample and determine elution volume
There is an inverse logarithmic relationship between the size of the molecule and the volume eluted
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Application of SEC
bull Protein separation and purification
bull Molecular weight determination
bull Determination of Oligomeric state (dimertrimer etc)
bull Protein-Protein interaction
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Excercise
bull 10 20 40 50 kDa protein
bull Which will elute first in gel filtration
bull Which will elute last in gel filtration
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
HIC (Hydrophobic interaction chromatography)
Principle bull Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an uncharged gel matrix
bull Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix
bull How is this achieved
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
General concept 1048713 Salt-promoted adsorption
HIC
bullHydrophobic groups on gel matrix and soluble proteins are shielded by water molecules
bullTo expose these hydrophobic regions water must be removed
bullthis can be achieved by adding high salt concentration in buffer (ammonium sulfate)
Protein
H2O
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
HIC
Protein
H2O
Protein in buffer containing High salt concentration (Hydrophobic patches on protein exposed)
Protein
H2O
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Protein
H2O
To elute bound protein= buffer containing no or low salt concentration or water only (Hydrophobic patches get buried inside protein)
Protein
H2O
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl
Butyl Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt
HIC
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Salting out Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations
The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
This process is also used to concentrate dilute solutions of proteins
Dialysis can be used to remove the salt if needed
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Principle of salting out
bull There are hydrophobic amino acids and hydrophilic amino acids in protein molecules
bull After protein folding in aqueous solution hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules
bull If enough of the protein surface is hydrophilic the protein can be dissolved in water
bull When the salt concentration is increased some of the water molecules are attracted by the salt ions which decreases the number of water molecules available to interact with the charged part of the protein
bull As a result of the increased demand for solvent molecules the protein-protein interactions are stronger than the solvent-solute interactions the protein molecules coagulate by forming hydrophobic interactions with each other This process is known as salting out
bull As different proteins have different compositions of amino acids different protein molecules precipitate at different concentrations of salt solution
Ammonium sulfate precipitation
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
What follows salting out step
bull Need to remove salt
bull Following a salting-out step the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps
bull Dialysis
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Dialysis
bull Only those molecules that are small enough to fit through the membrane pores are able move through the membrane and reach equilibrium with the entire volume of solution in the system
bull By contrast large molecules that cannot pass through the membrane pores will remain on the same side of the membrane as they were when dialysis was initiated
bull To remove additional unwanted substance it is necessary to replace the dialysis buffer so that a new concentration gradient can be established
bull Once the buffer is changed movement of particles from high (inside the membrane) to low (outside the membrane) concentration will resume until equilibrium is once again reached
Movement of molecules by diffusion from high concentration to low concentration through a
semi-permeable membrane
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Separation based on SIZE Dialysis
bull for separating proteins from small molecules amp ions Small molecules (blue) (buffer ions small organic molecules salt etc) pass through membrane diffusing into surrounding medium
bull Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --gt serial dilution getting rid of unwanted small molecules and ions
bull Dialysis membranes with different pore sizes available
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size
Factors Affecting Dialysis Rate
Factors that affect the completeness of dialysis include
(1) dialysis buffer volume
(2) buffer composition
(3) the number of buffer changes
(4) time
(5) temperature and
(6) particle size vs membrane pore size