What is affinity chromatography ?

Affinity chromatography technique exploits the property of the biomolecules such as enzymes to bind specifically to the substrate or any functional group[1].

Examples:

1. Enzyme binding to the substrate.
2. Antibody with specific Antigen.
3. Hormone binding to receptor.

Requirements:

1. Ligands which has an affinity for the substrate.
2. Matrix to hold the ligand.
3. The substrate which is your molecule of interest.
4. Allosteric Binding site.
5. Single step Purification.
6. Spacer arms so as to create space between the ligand and the matrix. If the ligand and matrix are close to each other, then due to steric hindrance, the molecule of interest would not be able to bind the allosteric binding site on the ligand.

Properties of Matrix:

• The matrix should be inert and stable. Its property should not change due to change in pH, Ionic strength and temperature during the elution technique.
• The matrix should have multiple binding sites for the Ligands. Thus, more the ligand binds the matrix, greater the resolution.
• The matrix should be porous so that surface area increase.
• Example of matrices are sepharose, agarose, etc.

Properties of Ligands:

• The ligand should have good affinity and specificity for the molecules of interest.
• The ligand should bind the molecule of interest in reversible fashion. If the molecule binds to the ligand with a very high affinity, then the molecules will not be eluted out.
• The ligand should have two binding sites. One to bind with the matrix and the other to bind with the molecule of interest.
• The two binding sites should not overlap. If so, then spacer arms should be used to attach matrix and ligand. Spacer arms such as Diaminopropanol are commonly used[2].
• Examples of ligands: Heparin ligands are used to isolate the fibronectins.