Lab Experiment 29.1 : Generation of Affinity Column for antibody purification

Generation of Receptor- The receptor molecule present on the matrix can be produced either by genetic engineering, isolation from the crude extract or in the case of antibody, it is produced in the mouse/rabbit model and purify. The generation of receptor molecule is beyond the discussion in the current lecture and interested student are advised to follow it from other relevant course.

Coupling of the Receptor- Once the receptor molecule is available, it can be couple to the matrix by following steps. (1) Matrix activation (2) covalent coupling utilizing reactive group on ligand. (3) deactivation of the remaining active group on matrix.

CNBr mediated receptor coupling- CNBr mediated coupling is more suitable for protein/peptide to the polysaccharide matrix such as agarose or dextran. CNBr reacts with polysaccharide at pH 11-12 to form reactive cynate ester with matrix or less reactive cyclic imidocarbonate group. Under alkaline condition these cynogen ester reacts with the amine group on receptor to form isourea derivative (Figure 29.1). The amount of cynate ester is more with agarose whereas imidacarbonate is more formed with dextran as a matrix. The protein or peptide ligand with free amino group is added to the activated matrix to couple the receptor for affinity purification.

 

Figure 29.1: CNBr mediated coupling of receptor to the matrix.