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:
- 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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