Affinity Chromatography - Lesson Summary
Affinity Chromatography works on the principle of mutual recognition forces between a ligand and a receptor.
Mutual interaction between a ligand and a receptor forms a ligand-receptor complex.
In the Bio-affinity Chromatography method, biomolecules are used as a receptor present on the matrix and it exploits the biological phenomenon such as the antibody-antigen.
The major determinants that are responsible for providing specificity in Affinity Chromatography are:
The epichlorohydrin activates the polysaccharide matrix by adding the oxirane group that contains a three-carbon alcohol group spacer arm.
Glutathione S-transferase (GST) utilizes glutathione as a substrate to catalyze conjugation reactions for xenobiotic detoxification purposes.
The GST fusion protein is produced by the recombining of the protein of interest with the GST sequence present in the expression vector.
The purified fusion can be treated with the thrombin to remove the GST tag from the protein of interest.
The Affinity Column can also be used as a tool to study or isolate an interacting partner of a particular protein.
The steps in the Ni-NTA Affinity Chromatography process are:
Regeneration of column
The Avidin-biotin System is used to capture and isolate cytokines from immune cells.
The Biotinylation of antibodies allows the immobilization of antibodies in the correct orientation on the streptomycin coated glass beads.