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AFFINITY CHROMATOGRAPHY

INTRODUCTION
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.

The high selectivity of affinity chromatography is caused by allowing the desired


molecule to interact with the stationary phase and be bound within the column in
order to be separated from the undesired material which will not interact and
elute first. The molecules no longer needed are first washed away with a buffer
while the desired proteins are let go in the presence of the eluting solvent (of
higher salt concentration).

PRINCIPLE

The principle of affinity chromatography is as follows:


1) Inject a sample into an initially equilibrated affinity chromatography
column.
2) Only the substances with affinity for the ligand are retained in the column.
3) Other substances with no affinity for the ligand are eluted from the column.
4) The substances retained in the column can be eluted from the column by
changing pH or salt or organic solvent concentration of the eluent.

TERMS USED:
Matrix: used for ligand attachment. Matrix should be chemically and physically
inert.
Spacer arm: used to improve binding between ligand and target molecule by
overcoming any effects of steric hindrance.
Ligand: molecule that binds reversibly to a specific target molecule or group of
target molecules.

WORKING CONDITION
The first step towards a successful purification is to determine the availability
of a suitable ligand that interacts reversibly with the target molecule or group of
molecules.
Affinity purification generally involves the following steps:
1. Incubate crude sample (e.g., cell lysate or serum) with the affinity support
to allow the target molecule in the sample to bind to the immobilized
ligand.
2. Wash away non-bound sample components from the support using
appropriate buffers that maintain the binding interaction between target
and ligand.
3. Elute (dissociate and recover) the target molecule from the immobilized
ligand by altering the buffer conditions so that the binding interaction no
longer occurs.

APPLICATION
Lectin Affinity Chromatography:
They can separate polysaccharides, glycopeptides, and oligosaccharides and cells
that contain particular carbohydrate structures.
Dye-Ligand Affinity Chromatography:
This is a method used to purify blood proteins, protein pharmaceutical agents,
some enzymes, and albumin.
Immunoaffinity Chromatography:
In this process, molecules such as peptides, viruses, hormones, and enzymes can
be purified in a column containing antibodies or other similar agents. Agarose or
HPAC can be used as the support.
Analytical Affinity Chromatography:
This method is used to study the reactions between biomolecules with lectins,
antibodies, and aptamers or drug binding with agents such as enzymes, serum
proteins, and receptors.