SRTARTING WITH THE NAME OF ALLAH THE MOST MERCIFUL AND MIGHTY

AFFINITY CHROMATOGRAPHY

DEFINITION
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 chromatographic matrix.

Affinity Chromatography is a relatively simple, yet quite effective molecular technique which isolates antibodies, antigens, hormones, or other proteins by taking advantage of their binding affinity for their respective ligands

Importance of affinity chromatography

Purification that would otherwise be timeconsuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography Target protein(s) is collected in a purified, concentrated form.

Common terms in affinity chromatography

Matrix

Spacer arm

Ligand

MATRIX
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.

LIGANDS
Molecule that binds reversibly to a specific target molecule or group of target molecules.

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.

 Affinity chromatography is widely used as a means of separation and purification with specific properties.

Affinity chromatography in practice

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.

Preparation of media and buffers

Storage solutions and preservatives should be washed away thoroughly before using any affinity medium.

Use high quality water and chemicals.

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Solutions should be filtered through 0.45 m or 0.22 m filters.

Reuse of affinity media depends on the nature of the sample and should only be performed with identical samples to prevent cross contamination.

Avoid using magnetic stirrers as they may damage the matrix. Use mild rotation or end-over-end stirring.