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Beer-Lambert Law

Beer-Lambert Law:

log(I0/I) = ebc

e = A/cb

A = ebc

A = ec (when b is 1 cm)

I0 = intensity of incident light

I = intensity of transmitted light

e = molar absoptivity coefficient in cm2

mol-1

c = concentration in mol L-1

b = pathlength of absorbing

solution in cm-1

A = absorbance = log(Io/I)

0.1

cm

ℓ

http://www.hellma-worldwide.de/en/default.asp

For example…

• Protein abs @ 280 (1g/L) = 1.0

• Abs is 0.67

• Thus – Abs/abs(1g/L)=0.67mg/ml

Protein purification and characterization

Exercise #5

Affinity Chromatography

Protein Purification & Characterization

What is Chromatography?Invented by a Russian botanist named Mikhail Tswett in 1905. He separated plant pigments using glass columns packed with calcium carbonate.

Before we proceed…

FPLC (AKTA Purifier) Gravity-assisted glass column

How we conduct chromatography?

עקרונות ההפרדהAffinity Chromatography

Affinity chromatography separates proteins on the basis of a reversible

interaction between a protein (or group of proteins) and a specific ligand

coupled to a chromatography matrix.

•What are the advantages of this technique?

•What’s the importance of the reversible binding?

Customization of affinity column

• Choosing the right ligand (verify dissociation)

• Choose the right matrix:

– Hydrophilic

– Large spaces

– Rigid

– Inert

– Chemically stable

• Attaching ligand to matrix

For example, immobilization of IgG to agarose beads

Aldehyde

activation

Choice of ligand

• Enzyme ↔ substrate analogue, inhibitor, cofactor.

• Antibody ↔ antigen, virus, cell.

• Lectin ↔ polysaccharide, glycoprotein, cell surface receptor, cell.

• Nucleic acid ↔ complementary base sequence, histones, nucleic

acid polymerase, nucleic acid binding protein.

• Hormone, vitamin ↔ receptor, carrier protein.

?

The dissociation constant (kD) for the ligand - target complex should ideally be in the

range 10-4 (enzyme and a weak inhibitor) to 10-8 M (hormone and hormone receptor) in

free solution.

Choice of a Matrix• Hydrophilic – to avoid non-specific interactions

• Large spaces – to increase surface area and enable large protein to enter

• Rigidity – to withstand the pressure of the solvent

• Inert – so it won’t affect the binding between the protein and the ligand

• Chemical stability

Method 1:The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand.

pH elutionA change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation.

Ionic strength elutionThe exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a linear gradient or in steps.

Methods for protein’s elution

Method 2:Extremes of pH or high concentrations of chaotropic agents are required for elution, but these may cause permanent or temporary damage.

Chaotropic eluentsIf other elution methods fail, deforming buffers, which alter the structure of proteins, canbe used, e.g. chaotropic agents such as guanidine hydrochloride or urea. Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein.

Methods for protein’s elution

Methods 3 and 4:Specific elution by addition of a substance that competes for binding. These methods can enhance the specificity of media that use group-specific ligands.

Competitive elutionSelective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand.

Methods for protein’s elution

And if our protein doesn’t have a known ligand?

Fusion proteins and tags

His tag

Kd coefficient

AC purification steps

1. Equilibrate column for the specific purification process.

2. Load and bind protein.

3. Wash the column 5-10 CV

4. Elute.

Equilibrate column for the specific purification process

20 mM TRIS pH 7.5

200 mM NaCl

10-40mM Imidazole

Similar to

Your lysis buffer

Loading column with protein solution

Protein sample is loaded and

only protein containing the

tag/binding site will be bound

to the column. The rest of

the proteins will elute as soon

as the sample is injected

20 mM TRIS pH 7.5

200-300 mM NaCl

10-40mM imidazole (trial&error)*

Your lysis buffer+protein

Column wash

Washing removes non-

specific binders and low

binding proteins.

20 mM TRIS pH 7.5

200-1000 mM NaCl

20-100 mM Imidazole

Elution

בשלב זה אנו מזרימים דרך

אנו . הקולונה את בופר האלוציה

משחררים את החלבון הקשור על

. ידי הוספת ריכוז גבוה של הליגנד

בשלב זה יש לאסוף דוגמאות על

מנת לזהות את החלבון מאוחר

.יותר

20 mM TRIS pH 7.5

50-200 mM NaCl

300-500mM Imidazole

GST-Glutathione S-transferase

26.4 kDa

GST-Glutathione S-transferase

Protocol

1. Add 5 ml of a 50:50 slurry of glutathione-Sepharose beads to a lysate of 5gr of cells pellet (assuming expression is medium). Buffer: PBS or equivalent supplemented with 5 mM of BME or DTT (2mM TCEP).

2. Incubate 30’ at 4C rotating.

3. Collect flow through.

4. Elute in the presence of freshly prepared 10-20mM reduced glutathione.

5. If yield is low you might try increasing to 50 mM of reduced glutathione

Maltose binding protein (MBP)

40.6 kDa

Cross linked Amylose beads

Elution with 10mM Maltose