The Protein Man's Blog | A Discussion of Protein Research

Commercially available affinity purification supports are designed on the principle of specific surface interactions among biomolecules, including, but not limiting to, antigen-antibody, enzyme-ligand and lectin-carbohydate. Affinity chromatography is one of the most efficient tools used for purification of biomolecules of interest, including proteins, glycoproteins, lipids and nucleic acids. In affinity chromatography, one of the interacting molecules is covalently bound to the resin and is addressed as a ligand. The stationary ligand bound resin interacts with the ligand interacting proteins or biomolecules, which are passed through the resin in mobile phase and hold them to the resin. These molecules are later eluted as a purified fraction with an elution buffer.

Affinity chromatography is the most popular and widely used method for purification of biomolecules including proteins, antibodies, lectins, carbohydrates, nucleic acids and glycoproteins. Affinity purification is based upon specific surface interactions among biological molecules such as antigen-antibody, enzyme-ligand etc. These specific surface interactions enable the purification of molecules from 1000 to 10,000 fold in a single step. In affinity chromatography, the ligand, antigen or one of the interacting molecules is covalently bound to the matrix and is used as a bait to fish out the target protein from any complex pool of proteins including cell or tissue lysate.

Proteins are highly heterogeneous, complex bio macromolecules consisting of one or more long chains of amino acids. Proteins or peptides fold up to form secondary and tertiary structures, and associate with other protein subunits to form quaternary structures. Proteins are structurally and functionally different from each other and require distinct surrounding environment for their stability and activity. Proteins are susceptible to degradation, denaturation and precipitation when taken out of their native environment.

Carbohydrate affinity chromatography is a method of choice for purification of glycoproteins, lectins and other carbohydrate metabolite proteins. This affinity chromatography uses carbohydrate ligands such as carbohydrates, glycoproteins and carbohydrate matrices for purification. The matrix needs to be activated for covalent immobilization of the ligand. Various methods used for the coupling of ligands depending upon the activating reagents are as follows: