Lab Experiment 29.2 : Purification of monoclonal antibody produced from the hydridoma cell lines using affinity chromatography.

Operation of the Affinity chromatography- Different steps in affinity chromatography is given in Figure 29.2

1. Equlibration- Affinity column material packed in a column and equilibrate with a buffer containing high salt (0.5M NaCl) to reduce the non-specific interaction of protein with the analyte.

2. Sample Preparation- The sample is prepared in the mobile phase and it should be free of suspended particle to avoid clogging of the column. The most recommended method to apply the sample is to inject the sample with a syringe. Load the supernatant from the hybridoma cell culture onto the column. Sample can reload onto the column 2-3 times to ensure 100% binding.

3. Wash the column 2 times with 10 colum volume using the equilibration buffer.

4. Elution- There are many ways to elute a analyte from the affinity column. (1) increasing concentration of counter ligand, (2) changing the pH polarity of the mobile phase, (3) By a detergent or chaotrophic salt to partially denature the receptor to reduce the affinity for bound ligand.

5. Neutrilize the acidic elute with 1M Tris pH 7.2 containing 150mM NaCl. The purified antigen can be stoed at -20⁰C.

6. Column Regeneration- After the elution of analyte, affinity column requires a regeneration step to use next time. column is washed with 6M urea or guanidine hydrochloride to remove all non-specifically bound protein. The column is then equiliberated with mobile phase to regenerate the column. The column can be store at 4⁰C in the presence of 20% alchol containing 0.05% sodium azide.