AFFINITY

CHROMATOGRAPHY
VARIOUS BIOMOLECULE PURIFICATION TECHNIQUES
 INTRODUCTION
Affinity Chromatography is essentially a sample
purification technique, used primarily for biological
molecules such as proteins.

It is based on the principle of specific interaction

between the protein or antigen and antibody for
separation of biomolecules

It is a method of separating a mixture of proteins or

nucleic acids molecules by specific interactions of
those molecules with a component known as a ligand,
which is immobilized on a support (SP).
 INTRODUCTION
If a solution of, say, a mixture of proteins is passed
over (through) the column, one of the proteins binds
to the ligand on the basis of specificity and high
affinity (they fit together like a lock and key).

The other proteins in the solution wash through the

column because they were not able to bind to the
ligand.
 INTRODUCTION
Affinity chromatography is used for isolation of all biological
macromolecules

It is used to purify nucleic acid, antibodies, enzymes, etc

To determine the binding of biological compound to a particular

substance
 PRINCIPLE
Affinity chromatography is one of the most diverse and
powerful chromatographic methods for purification of
a specific molecule or a group of molecules from
complex mixtures.

It is based on highly specific biological interactions

between two molecules such as interactions between
enzyme and substrate, receptor and ligand, or
antibody and antigen.
 PRINCIPLE
These interactions which are typically reversible are
used for purification by placing one of the interacting
molecules referred to as affinity ligand onto a solid
matrix to create a stationary phase while a target
molecule is in the mobile phase.

Many of the commonly used ligands coupled to affinity

matrices are now commercially available and are
ready to use.
 CHROMATOGRAPHIC MEDIA
A matrix in its use here is a substance, usually in
bead form to which a specific ligand is covalently
bound.

In order for the matrix to be effective, it must have

certain characters:

1)It must be insoluble in solvents and buffers

employed in the process

2)It must be chemically and mechanically stable

 CHROMATOGRAPHIC MEDIA
3)It must be easily coupled to a ligand or spacer arm
onto which the ligand can be attached.

4)It must exhibit good flow properties and have a

relatively large surface area for attachment
 IMMOBILISED LIGAND
The ligand can be selected only after the nature of the
macromolecule to be isolated is known.

• When a hormone receptor protein is to be purified by

affinity chromatography, the hormone itself is an ideal
candidate for the ligand.

• For antibody isolation, an antigen or hapten may be used

as ligand.

• If an enzyme is to be purified, a substrate analog,

inhibitor, cofactor, or effector may be used as a the
immobilized ligand.
 ATTACHMENT OF LIGAND TO MATRIX
Several procedures have been developed for the covalent
attachment of the ligand to the stationary phase. All
procedures for gel modification proceed in two separate
chemical steps:

1)Activation of the functional groups on the matrix and

2)Joining of the ligand to the functional group on the matrix.

A wide variety of activated gels is now commercially

available. The most widely used are given below -
 ATTACHMENT OF LIGAND TO MATRIX
 Cyanogen bromide-activated agarose (attachment of ligand
containing10amino group)

 6-aminohexanoic acid-agarose (Overcomes steric

interference by positioning spacer arm )

 1,6-diaminohexane-agarose (Overcomes steric

interference by positioning spacer arm )

 Carbonyldimidazole (cdi)-activated supports

(produces uncharged N-alkylcarbamate groups)

 ATTACHMENT OF LIGAND TO MATRIX
 Epoxy-activated agarose (attachment of ligands
containing hydroxyl, thiol, or amino groups)

 Group specific adsorbents (contains ligands that

have affinity for a class of biochemically related
substances)
 EXPERIMENTAL PROCEDURE
1. SELECT GEL (MATRIX) AND LIGAND

2. COUPLE LIGAND WITH MATRIX OR GEL

3. PREPARE GEL FOR COLUMN

4. PACK GEL IN GLASS COLUMN

5. SET-UP COLUMN EQUIPMENT

6. EQUILIBERATE COLUMN WITH BUFFER

7. APPLY SAMPLE
 EXPERIMENTAL PROCEDURE
8. WASH COLUMN TO REMOVE UNBOUND MOLECULES

9. ELUTE BOUND MOLECULES

10. COLLECT AND ANALYZE ELUENT

11. REGENERATE AND STORE GEL

17
 EXPERIMENTAL PROCEDURE
SELECTION OF A GEL OR LIGAND - Many type of
matrix-ligand systems are commercially available and
cost are reasonable so time can be saved by purchasing
preactivated gel for direct attachment of ligand.
 SPACER ARM
The binding site of a target protein is often located
deep within the molecule and an affinity medium
prepared by coupling small ligands, directly to matrix
may exhibit low binding capacity due to steric
interference i.e. the ligand is unable to access the
binding site of the target molecule.

This happens specially with the smaller ligands.

In this case, we need to select a pre-activated matrix

with a spacer arm.
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 EXPERIMENTAL PROCEDURE
BUFFER - Binding buffer is used for formation of complex
between a matrix and ligand as slight change in ionic
concentration weakens the interactions between them.

WASHING – This is done after application of sample

Molecules that bind to the ligand will remain associated with

the stationary phase.

A wash buffer is then applied to remove non-target

biomolecules by disrupting their weaker interactions with the
stationary phase, while the biomolecules of interest will
remain bound.
 EXPERIMENTAL PROCEDURE
AFFINITY ELUTION – This is used for removal of target
molecules. In the process of affinity elution, target
biomolecules are removed by applying a so-called
elution buffer, which disrupts interactions between the
bound target biomolecules and the ligand.

Sometimes, selective substances are added to the

elution buffer which causes selective elution of bound
macromolecule-ligand complex, resulting in elution of
desired macromolecule.
 EXPERIMENTAL PROCEDURE
CHAOTROPIC AGENTS - If gentle and selective elution
methods do not release the bound macromolecule then
mild denaturing agents can be added to the buffer.

The most powerful agents are urea and guanidine.

 APPLICATIONS
It is used for isolation and purification of all biological
macromolecule.

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.
 APPLICATIONS
Purification that would otherwise be time-consuming,
difficult or even impossible using other techniques can
often be easily achieved with affinity chromatography.

It is used to purify nucleic acid, antibodies, enzymes

etc.,

To determine which biological compound bind to a

particular substance.

To reduce amount of substance in a mixture