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Affinity Chromatography


Affinity chromatography is one of the most effective methods used for the purification of proteins. As with the ion exchange chromatography, the separation of compounds is based on the characteristics of many analyte to enter into specific interaction with certain molecules of the packed material. An  electrostatic forces between charged groups, nonpolar interactions, hydrogen bonds, hydrophobic bonds may be involved in addition to covalent bonding through the use of  biospecific ligands (e.g. enzyme substrates, antibodies, receptors) or  so-called pseudo-biospecific ligands (e.g. lectines, dyes, sulfur containing groups, etc.) are involved in the interaction process (see Fig below).   

Affinity Chromatography: Principle

Separation of compounds by affinity chromatography requires the following steps:

  1. Preparation of the affinity column: a biospecific ligand is covalently attached to a chromatographic bed material called a matrix.

Can you guess the various factors that influence the selection of appropriate ligand?

2.      Sample adsorption with ligand in the column

3.      Washing of impurities.

4.      Removal of bound sample with ‘specific’ or ‘nonspecific’ elution methods.

Can you suggest how you could elute and isolate  a specific  antibody for functional studies through this mode of chromatography?

Could you design a different elution system if you were isolating the antibody for amino acid sequencing?

Give examples of substances that may be isolated through this mode of chromatography

Note on packing material: sepharose, a bead-formed from agarose gel, is the most commonly used matrix for immobilizing biologically active molecules. The hydroxyl groups on the sugar residues can be easily derivatised for covalent attachment of a ligand.

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