[toc wrapping=left]
Affinity chromatography operating principle
https://www.youtube.com/watch?v=FUAQKjKT99Y
Separation of molecules by affinity chromatography (AC) relies on differences in affinity between solute molecules and specific ligand sites on an insoluble, inert matrix. For example, an enzyme might preferentially bind to ligand sites on the matrix that mimic the natural substrate of the enzyme. Solutes that do not have substrate-binding sites that are structurally related to the matrix ligand sites will be poorly bound, if at all, to the matrix.
The legend below will identify the salient features of the diagram above. The spacer arm, which can have various lengths, allows the binding site to project outwards from the matrix, facilitating access to the complementary sites on the solute
Affinity chromatography applications
Affinity chromatography is a powerful technique, which can be used with a variety of biomolecules. One-step purification is not uncommon, and routinely, purification of the order of 100 to 1000-fold are easily achieved. The following table lists the major types of application:
Biomolecule |
Ligand type on matrix |
|---|---|
| Enzymes | Substrate, inhibitor, cofactor |
| Antibodies | Antigen, virus, cell |
| Nucleic acids | Complementary base sequence, histone, nucleic acid, polymerase, binding proteins |
| Hormones | Receptor, carrier protein |
| Lectins | Polysaccharide, glycoprotein, cell receptor |