Protein A Purification of Antibody

Reagents

1. Affi-gel Protein-A Agarose (BioRad #153-6153)
2. MAPS II Binding Buffer (BioRad # 153-6161)
   0.314 g/ml diH₂O
3. MAPS II Elution Buffer (BioRad #153-6162)
   0.023 g/ml diH₂O
4. MAPS II Regeneration Buffer (BioRad #153-6166
5. 1M Tris, pH 9-11
6. PBS

Procedure

1. Dilute Ab 1:5 with Binding Buffer. Filter through a 0.22 m filter.
2. Equilibrate column with at least 2 bed volumes of Binding Buffer
3. Apply Sample.
4. Wash column with 5-6 bed volumes of Binding Buffer. Collect 1 ml fractions until base line is achieved.
5. Elute Ab with 5 bed volumes of Elution Buffer. Collect 1 ml fractions into 50 ml 1M Tris, until base line is achieved.
6. Take 50 ml aliquots of each fraction for ELISA.
7. Pool fractions containing Ab. Dialyze against 2 x 4L PBS, overnight, 2-8^oC.
8. Re-equilibrate column with Binding Buffer. Store column at 2-8^oC.
9. Determine protein concentration of Ab, aliquot, and store at -20^oC.

Note

Extinction Coefficient for IgG = 1.4 and for IgM = 1.2