5/24/2022

Affinity Chromatography

Affinity Chromatography
Method of separating a component from
complex mixture based on a highly specific (but
reversible) interaction between antigen and
antibody, enzyme and substrate,
receptor and ligand, or protein and nucleic acid
etc.

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The resin

Affinity Ligand

Matrix

Examples of tags and ligands

• His-tag • Transition metal ion
• FLAGTM peptide • Monoclonal antibody
• Strep-tag • Biotin
• GST tag • Glutathione
• Maltose binding protein fusion • Amylose
• Calmodulin binding protein fusion • Ca2+

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Step 1. Loading affinity column.

Step 2. Mixture of proteins sieve through matrix of affinity beads.

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Step 3. Proteins interact with affinity ligand with

some binding loosely and others tightly.

Step 4. Wash off proteins that do not bind.

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Step 5. Wash off proteins that bind loosely.

Step 6. Elute proteins that bind tightly to ligand and collect

purified protein of interest.

The kinds of Elution

pH Elution
Ionic Strength Elution
Reduced Polarity of Eluent
Competitive Elution
Chemotropic Eluents

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Affinity chromatography applied to recombinant proteins

Purity test
SDS-PAGE

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Affinity chromatography

The technique offers high selectivity, high resolution, and usually

high capacity for the proteins/biomolecules of interest.

Purification that would otherwise be time-consuming, difficult or

even impossible using other techniques can often be easily
achieved with affinity chromatography.

The technique can be used to separate active biomolecules from

denatured or functionally different forms, to isolate pure
substances present at low concentration in large volumes of crude
sample and also to remove specific contaminants.

Gel Filtration
Chromatography

Gel Permeation Chromatography

Size Exclusion Chromatography
Molecular Sieve Chromatography

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Gel Filtration Chromatography

• Gel filtration chromatography separates

molecules according to their size and
shape.

• The stationary phase consists of beads

containing pores that span a relatively
narrow size range.

Smaller molecules spend more time inside

the beads than larger molecules and
therefore elute later (after a larger volume of
mobile phase has passed through the
column).

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Types of gels used

• The gels used as molecular sieves are
cross linked polymers.

• They are uncharged and inert i.e. don’t

bind or react with the materials being
analyzed.

Example
Dextran: is a homopolysaccharide of glucose
residues.

• It’s prepared with various degrees of cross-

linking to control pore size.

• It’s bought as dry beads, the beads swell when

water is added.

• The trade name is sephadex.

• It’s mainly used for separation of small peptides

and globular proteins with small to average
molecular mass.

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Picture from Amersham website

Pores in the gel

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Advantages of Gel filtration

It’s the one of the best methods for separation of
molecules differing in molecular weight because:

1. It doesn’t depend on temperature, pH, ionic

strength and buffer composition. So separation
can be carried out under any conditions.

2. There is very little adsorption

3. The elution volume is related to the molecular

weight

Applications of gel filtration

• Purification of enzymes and other proteins.

• Estimation of molecular weight mainly for

globular proteins:

• Fractionation of same class of

biomolecules according to size

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