Affinity/purification process

Avidin is a tetrameric protein deposited in the egg white of birds, reptile and amphibians. This protein has affinity for biotin, cofactor of several enzymes. Immobilized avidin column is used for purification of biotin containing enzyme. N on-biotin molecules do not bind to immobilized avidin and are washed away. Bound proteins with biotin may be competitively eluted using 2 mM biotin.

Calmodulin, a regulatory Ca⁺² binding protein, is present in all eukaryotic cells. Calmodulin binds proteins through their interactions with hydrophobic sites on its surface which is exposed after Ca⁺² binding to the enzyme. Elustion is done by chelating agent such as EGTA or EDTA. Once Ca⁺² bound to Calmodulin is chelated, a reversal of the conformational change which expose the protein binding sites takes place. This results in protein elution.

Concanavalin A is tetrameric metalloprotein. This protein is a carbohydrate-binding protein (lectin) originally lycoproteins containing a-D-mannopyanosyl and a-D-glucopyranosyl residues. Elusion of bound glycoprotein is achieved by increasing gradient of α-D- methylmannoside or α -D- methylglucoside.

Cibacron Blue F3G-A is a sulfonated triazine dye that can be immobilized on a solid support and used for affinity chromatography of nucleotide-requiring enzymes.