ex. 6 and 7

Exercise 6

Gel Chromatograph

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OBJECTIVE

Operate a gel filtration column using colored mixtures

Calculate the parameters involved in gel filtration chromatography

Determine the molecular weight of the albumin isolate

CHROMATOGRAPHY

collective term for a family of laboratory techniques for the separation of mixtures

is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary phase while the other the mobile phase moves in a definite direction.

GEL CHROMATOGRAPHY

is a chromatographic method in which molecules in solution are separated based on their size

Also recognized as: gel permeation chromatography size exclusion chromatography molecular sieve chromatography gel filtration chromatography

GEL CHROMATOGRAPHY

Application and Usage

purification determination of the molecular weight

of the protein removing of low molecular weight

impurities (desalting)

SIZE EXCLUSION CHROMATOGRAPHY

developed in the 1950s using cross-linked dextrans

involves a partition of molecules between two liquid volumes: the volume of the mobile phase the accessible volume contained within the

stationary porous bead


Proteins can be separated in their native conformation since there is no interaction with the resin there is good recovery of biological activity

disadvantage it has low capacity, will dilute the sample

applied and shows at best moderate resolution


named ‘size exclusion’ chromatography because the pores within the material have a

maximum pore size

molecules larger than the pore size will be excluded from entering into the porous material and will travel down the column in the liquid volume surrounding the porous beads

small molecules will also be able to travel down the column in the liquid volume surrounding the porous beads but they will also be able to diffuse into the volume within the beads

GEL CHROMATOGRAPHY

Principle:

GEL CHROMATOGRAPHY

The mode of action can be simplified as:

“Larger molecules are excluded and migrate faster and smaller molecules are included and retained longer.”

EXPERIMENT PROPER

Preparation of the gel filtration media

Desired Characteristics: (1) chemical inertness of the gel

matrix,(2) low content of ionic groups (3) uniform pore and particle size(4) wide choice of gel particle and pore

size(5) high mechanical rigidity.

VARIOUS GEL FILTRATION MATRICES

Gel filtration matrices are generally cross-linked products of dextran, agarose, and polyacrylamide

DEXTRAN

One of the most widely used matrices in gel filtration is Sephadex

It is a branched polysaccharide made of many glucose molecules joined into chains of varying lengths.


Sephadex gels

produced by permitting a microorganism, Leuconostoc mesenteroides, to ferment sucrose into large polymers of glucose

the polyglucose units are purified and cross-linked by treatment with epichlorohydrin into various classes of gel beads


A portion of Dextran polymer.

Sephadex gels

usually stable in water, salt solutions & organic solvents

buffers a wide pH range

but their glycosidic linkages can be hydrolyzed in the presence of strong acid


Sephadex gels

These gels are identified by a number such as G-10 or G-200, which refers to the water regain value of the gel multiplied by a factor of 10.

Designation Mesh Water Regain (mL/dry gel)

Sephadex G-10 1.0 + 0.1

Sephadex G-15 1.5 + 0.2

Sephadex G-25 CourseMedium

FineSuperfine

2.5 + 0.2

Sephadex G-50 CourseMedium

FineSuperfine

5.0 + 0.3

Sephadex G-75 Superfine 7.5 + 0.5

Sephadex G-100 Superfine 10 + 1.0


POLYACRYLAMIDE

produced by polymerizing acrylamide into bead form

usually identified by a number such as P-10 or P-100, which indicates the exclusion limit of the gel in thousands of Daltons


Large pored polydextran and polyacrilamide gels can be used to separate solute only up to

300,000 daltons may be difficult to use owing to their lack of

mechanical rigidity

During chromatography even slight pressure, including osmotic pressure, can cause distortion and irregular packing as a result, they tend to compress in the

column, causing unacceptably slow flow rates


AGAROSE for fractionation of large molecules

Sepharose is a bead-formed gel prepared from agarose

Gel stabilized by H-bonding NOT by covalent linkage


AGAROSE

Additionally, the presence of the unusual sugar 3,6-anhydro-L-galactose in Sepharose contributes to its resistance effect to biological degradation.

Although agarose gels differ in bead structure from polydextran gels, their partition coefficients are also a function of molecular size.


COMBINED POLYACRYLAMIDE AND AGAROSE GELS

claimed to yield resolution in the presence of large amounts of hydrogen bond breaking agents and to give substantially increased flow rates

Ex.Sephacryl is a rigid gel prepared by cross-

linking dextran with acrylamide.Ultragels are the cross-linked products of

agarose gels with acrylamide.


COMBINED POLYACRYLAMIDE AND AGAROSE GELS

Sephacryls and Ultragels confer greater rigidity and lower polyacrylamide allows high degree of

separation agarose maintains rigidity flowrates may be used


The types of matrices differ in their degree of cross-linking and hence in their fractionation range.

The G-types of Sephadex differ in the degree of their crosslinking and thus their fractionation range.

- Usually, the higher the number, the higher is the fractionation range.

Some matrices are available in different particle sizes.

- The highest resolution is obtained with the smallest particle size.


The types of matrices differ in their degree of cross-linking and hence in their fractionation range.

Operating pressure is highest with the smallest size or super fine particles.

- Large-size particles are generally used for preparative chromatography, where a high flow rate at a low operation pressure is essential.

SEPHADEX G-100SEPHADEX G-100FRACTION RANGE : 4000 – 150,000 Da

MW ≤ 150,000 SELECTIVELY ELUTED

MW > 150,000 SUPER ELUTED

MW ≤ 4,000 SUPER TRAPPED

BUFFER

choice of buffer depends on the nature and compatibility of the protein of

interest There are some considerations about

possible interactions between the packing materials and the protein molecules including the target molecule.

Buffers of the above ionic strength are suitable to avoid such interactions between the packing material and the protein molecules. presence of ionic interaction is shown by

“tailing”

COLUMN

The column acts as the system for filtration.

The design characteristics of a suitable column include:

(1) a small dead space (the space between the medium support and the outlet of the column

(2) facilities for attachment of capillary tubing to the effluent fitting of the column

(3) a gel bed support that cannot be easily clogged (nylon screening is often used here)

(4) some means of protecting the bed surface

COLUMN

The choice of the column size mainly depends on the amount of protein to be purified. increase in column length and diameter can tighten

the resolution of the chromatographic procedure and increase the capacity of the column, respectively

The column should also be properly packed to avoid collapse of the pores in the beads resulting

in a loss of resolution (overpacked column) to avoid reduction of the relative surface area of

the stationary phase that is accessible to smaller species which may result in those species spending less time trapped in pores

COLUMN

Column should be checked for the presence of air bubbles or uneven column packing

can be inspected visually with the help of a hand-held lamp

applying colored sample material of high molecular weight (above the exclusion limit) to the column Blue dextran (MW approximately 2 x 106) will

migrate through the column as an evenly flowing blue band when column materials are packed well. A streaking blue dextran band indicates uneven or poor packing. In such case, the column should be repoured and tested again.

COLUMN

SOME PARAMETERSSOME PARAMETERSVO = VOID VOLUME = volume of blue dextran

Vt = TOTAL VOLUME = Πr2h

Ve = ELUTION VOLUME

VI = INTERNAL VOLUME = VT - VO

Vol of sample = 1 - 5 % Vt = 0.01 - 0.05 Vt

Standards MW (Da)

Blue Dextran 2 x 106

BSA 66, 382

Myoglobin 17, 500

Bromphenol Blue 670

CALIBRATION

set standards that will help in estimating the molecular weight of the protein sample

ELUTION VOLUME (Ve)

1 2 3 4 5 6 7 8 9 10 11 12 13

3 mL min

Ve = vol. before colored tube + (½) vol. colored tubes

Ve BD = 2 (3 mL) + (½) (3) (3 mL) = 10.5 mL

Ve M = 7 (3 mL) + (½) (2) (3 mL) = 24 mL

Ve BCP = 11 (3 mL) + (½) (2) (3 mL) = 36 mL

ELUTION VOLUME (Ve)

Using elution profile

A5

00,

A6

50,

A5

40

Elution volume, Ve (mL)Ve A Ve sampleVe B Ve C

--- standards--- sample

CALIBRATION

Figure 6.1. Plot of absorbance vs. volume collected of the colored mixture and protein standards.

EGG ALBUMIN ISOLATE

Figure 6.2. Plot of absorbance vs. volume collected of different fractions collected after gel chromatography of egg albumin sample.

DETERMINATION OF MW OF ALBUMIN

Figure 6.3. Plot of absorbance vs. volume collected of the colored mixture and egg albumin isolate.

RESULTS

Figure 6.4. Plot of elution volume vs. log MW of the protein standards.

STANDARD MW (Da) log MW Ve (mL)

Blue dextran 2 x 106 6.3 5

BSA 66, 382 4.82 9

Myoglobin 65,000 4.81 11

Bromphenol blue 330 2.51 17

Albumin 1141020 6.0573 5.2

RESULTS

An partition coefficient (Kav) can be determined for proteins fractionated.

Ve is the elution volume of the sample of interest.

V0 is the void volume of the column. Vt is the total volume column.

PARTITION COEFFICIENT (KAV)


Solution Elution Volume (mL)

Void Volume (Vo), mL

Total Volume (Vt), mL

Kav

Blue dextran 55 7.065

0

BSA 9 1.937

Myoglobin 11 2.906

Bromphenol blue

17 5.811

Egg Albumin 5.2 0.097


= 0 no solute entered the pores due to MW

= 1 [solute]out = [solute]in

< 1 [solute]out > [solute]in Only few entered the beads

> 1 [solute]out < [solute]in More entered than freely eluted

GEL CHROMATOGRAPHY

Widespread application of gel chromatography results from its many advantages:

Gentleness of the technique permitting separation of labile molecular species

Solute recovery approaching 100 % High reproducibility A broad range of sample sizes Relatively short times and inexpensive

equipment needed for its performance.

APPLICATIONS

SEC is generally considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final "polishing" step of a purification.

The technique can determine the quaternary structure of purified proteins which have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions

APPLICATIONS

It is also helpful in the determination of the properties of the macromolecule such as strength and viscosity and it can also help in the evaluation of the presence of low molecular weight species that can serve as plasticizers, oligomers and monomers.

Exercise 7

Electrophoresis

Electrophoresis

Objectives:

•assemble the apparatus to be used for electrophoresis•prepare the gels as well as samples for electrophoretic analysis•load the sample in the gel•stain and destain the gel•determination of the molecular weight of the albumin using the SDS-PAGE result

Electrophoresis

Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.

Basis of separation:Molecular weight/ sizeNet charge of the molecule

TheoryIf a molecule of net charge q is placed in an electric field, a force F is exerted upon it, which depends on the charge possessed by the molecule and the strength of the field into which it is placed. This is expressed mathematically as

F = E (q) d

where E is the potential difference between the electrodes and d is the distance between them.

Theory• The pulling force of the electric field is opposed by the drag or friction occurring between the accelerating molecule and the solution.

• In electrophoresis, voltage and current are supplied by a DC (direct current) power supply, and the electrodes, buffer, and gel are considered to be resistors.

•Power supply is used to hold one electrical parameter (current, voltage, or power) constant.

Theory• Since each molecule is expected to possess a unique charge and size, it migrates to a unique position within the electric field in a given length of time.

•Therefore, if a mixture of proteins is subjected to electrophoresis each of the proteins would be expected to concentrate into a tight migrating band at unique positions in the electric field.

Mixture of macromolecules

Porous gel

electrophoresis

cathode

anode

GEL MEDIUMElectrophoresis of macromolecules is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix.

The matrix can be composed of a number of different materials including paper, cellulose, acetate, or gels made of starch, agarose, or polyacrylamide.

However, polyacrylamide is the most common matrix for separating proteins, probably due to its versatile applications.

Synthesis of acrylamide gelsThe compounds used to construct the polymer matrix are

Synthesis of acrylamide gels

Two catalysts are required to initiate the polymerization: TEMED and ammonium persulfate.

TEMED catalyzes the decomposition of the persulfate ion to produce a free radical:

S2O8-2 2SO4

-

Synthesis of acrylamide gels• If these free radicals are brought into contact with acrylamide, a reaction occurs, with the preservation of the free radical within the acrylamide molecule.

•This “activated” acrylamide can then react in the same way with successive acrylamide molecules to produce a long polymer chain.


A solution of these polymer chains, although viscous, does not form a gel.

No gelation occurs because the long chains can slide past one another. Gel formation requires hooking various chains together or cross-linking them to one another.


This is done by carrying out polymerization in the presence of N,N’-methyl-bis(acrylamide), a compound that can be thought of as two acrylamide molecules coupled head to head at their nonreactive ends.

Carrying out polymerization in this manner yields a net of acrylamide chains.


The result is a solid polyacrylamide gel which is comprised of a porous network of cross-linked acrylamide monomers resulting in closed loops and a complex “web” polymer.

Cathode/Anode Reactions

Reactions that permit current passage from the cathode to the anode are shown below:

Cathode reaction: 2e- + 2H2O 2 OH- + H2

HA + OH- A- + H2O Anode reaction: H2O 2H+ + 1/2 O2 + 2e-

H+ + A- HA

These reactions are essentially the electrolysis of water, producing hydrogen at the cathode and oxygen at the anode.

Cathode/Anode Reactions

Note that for every mole of hydrogen produced, only one-half mole of oxygen is produced.

This affords an easy way of checking the electrodes to make certain that they are operating with the desired polarity.

FACTORS AFFECTING RATE OF MIGRATION

NET CHARGE NET CHARGE

depends on pH of medium and IpH of the proteinnegatively charged proteins migrate to the anode at a faster ratepH usually set at 4.5 – 9.0


Ionic Strength Ionic Strength The buffer serves :

to maintain a constant pH within the reservoirs and within the acrylamide gel as the electrolyte which conducts current across the electric field.

[buffer], IS , diffuse bands, resolution

[buffer], “burned” samples


VOLTAGEVOLTAGE

Voltage, rate of migration

KE of molecules, rate of migration

TEMPERATURETEMPERATURE

SIZE OF PORESSIZE OF PORES

pore size, rate of migration, resolution

Frowning & Smiling Gels

The cause of frowning and smiling gels is because of uneven application of current or changing temperature throughout the running of the set-up.

The current in the solution between the electrodes is conducted mainly by the buffer ions with a small proportion being conducted by the sample ions.

If the current is not evenly distributed throughout the gel, it may cause samples in the gel to move at different speed and forms a frowning or smiling shape depending on what part of the gel does the sample moves faster.

Frowning & Smiling Gels

Temperature also affects rate of migration and if an uneven distribution of temperature is applied the sample will move through the gel at different rates causing the frowning or smiling of gels.

NATIVE PAGE

Used to determine purity

Since the proteins remain in the native state they may be visualized not only by general protein staining reagents but also by specific enzyme-linked staining.

The disadvantage of this kind of electrophoresis is that the basis of separation are charge, shape and size unlike SDS-PAGE where the basis is only size.

SDS PAGE

Separation is based on MW alone

Used to determine MW and number of sub-units, and purify proteins prior to sequencing

Same as with Native PAGE but with SDS and -mercaptoethanol

NATIVE PAGENATIVE PAGE SDS PAGESDS PAGE

NET (-) charge on proteinthus, separation is based only on MW

SDS & 2-mercaptoethanol

SDS & 2-mercaptoethanolDenatures proteinsDissociates protein into subunitsCompletely unfolds each polypeptide chain

-MERCAPTOETHANOL-MERCAPTOETHANOL

Breaks disulfide bonds

Long, rod-like SDS-polypeptide complex

Staining MethodAt pH 9, a commonly used pH for electrophoresis, most protein are negatively charged. When the power is turned on, the proteins start migrating toward the positively charged anode.

Often a “tracking dye” such as bromphenol blue is included in the sample as a reference.

This colored material migrates faster than any of the macromolecules.

Therefore, if electrophoresis is continued until the dye reaches the bottom of the tube, one can be reasonably sure that all the macromolecules are still within the gel.

Staining MethodThe most commonly used protein stain is Coomassie blue in a methanol–acetic acid mixture (used to precipitate the proteins within the gel, preventing them from floating away before analysis).

Silver staining increases the level of sensitivity approximately 100-fold, detecting down to 1 ng of protein in a band. Silver staining can be used instead of Coomassie blue (or after Coomassie blue staining) to increase the levels of detection.

Staining Method

A rapid method of visualizing polypeptide bands in gels is to include 0.5% (v/v) trichloroethanol (TCE) in the acrylamide mixture prior to polymerization.

After electrophoresis the polypeptides can be visualized by exposure to UV light for 5 min, which catalyzes a reaction between tryptophan and TCE to produce a fluorescent product.

Standards S A M P L E Standards

Tracking dye

MW DeterminationThe distance a polypeptide migrates in SDS-PAGE is inversely proportional to log MW.

It is common practice to load a series of standard proteins with known molecular masses on a polyacrylamide gel at the same time as the samples.

The relative migration distances of these standard polypeptides can be measured and plotted against the log of their MW. The calibration graph can then be used to estimate the molecular masses of polypeptides in the samples.

MW Determination

Log

MW

Rf

Rf = _ distance traveled by sample___ distance traveled by tracking dye


Tracking dye


Tracking dye

A

B

MW Determination

Figure 7.1. Plot of Rf vs log MW of the molecular mark.

MW DeterminationProtein

standardsMW

(kDa)log MW Distance

traveled by the sample (mm)

Distance traveled by the tracking

dye (mm)

Rf

1 151.1760

4953 0.9245

2 201.3010

410.7735

3 301.4771

360.6792

4 401.6020

310.5849

5 501.6989

240.4528

6 601.7781

190.3584

7 701.8450

160.3018

8 901.9542

130.2452

Egg albumin samples

(egg albumin extract, (NH4)2SO4 fraction, and fraction 9, 10,11 Gel chrom albumin isolate)

Albumin 67.15 1.8270 18 53 0.3396

ELECTROPHORESIS OF NUCLEIC ACID

It is also possible to use gel electrophoresis in separating nucleic acid mixtures by using capillary electrophoresis (CE).

This type of electrophoresis can separate a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, carbohydrates, peptides and proteins based on their sizes and ionic properties.

ELECTROPHORESIS OF NUCLEIC ACID

CE separations of proteins and peptides are based on charge-to-mass ratios. The capillaries are also thin-walled.

In CE, buffer flow is generated inside the column when the electric field is applied. This flow is from the cathode electrode to the anode electrode in a a fused-silica capillary column; this movement of the buffer is called the electroosmotic flow (EOF).

SDS-PAGE vs GEL CHROMATOGRAPHY

The difference between SDS-PAGE and gel filtration chromatography is that in SDS-PAGE, the smaller passes through the gel faster leaving the larger molecules in the upper portion of the gel, which is opposite to gel chromatography where the largest molecules moves the fastest.

ex. 6 and 7

Documents

gel filtration column

gel chromatography objective

gel matrix

rigid gel

beadformed gel

various classes of gel

wide choice of gel particle

agarose gels