Loading/Binding Phase: The sample is introduced into a column with immobilized ligand. The target molecule binds specifically, while non-target molecules pass through.
Washing Phase: Non-specifically bound molecules are removed using an appropriate buffer.
Elution Phase: The target molecule is released by disrupting the interaction, often by changing pH, ionic strength, or adding a competing ligand.
Regeneration Phase: The column is restored for reuse by washing away residual target molecules and elution agents.
Classification of Affinity Chromatography
Based on Type of Interaction:
Enzyme-Substrate/Inhibitor Chromatography: Enzymes attached to the resin separate substrates or inhibitors.
Antigen-Antibody Chromatography: Antibodies or antigens immobilized to capture their counterpart.
Receptor-Ligand Chromatography: Receptors immobilized to capture specific ligands.
Based on Elution Method:
Biospecific Elution: Uses agents that specifically interfere with target binding.
Non-specific Elution: Alters pH or ionic strength to disrupt all bound molecules.
Specialized Types:
Immunoaffinity Chromatography: Uses antibody-antigen interactions for high specificity.
Metal Chelate Chromatography: Captures proteins with affinity for certain metals.
Dye-Ligand Chromatography: Employs reactive dyes as ligands to bind specific proteins