Protein A Purification of Antibody

1. Reagents

(1) Affi-gel Protein-A Agarose (BioRad #153-6153)

(2) MAPS II Binding Buffer (BioRad # 153-6161)

(3) 0.314 g/ml diH2O

(4) MAPS II Elution Buffer (BioRad #153-6162)

(5) 0.023 g/ml diH2O

(6) MAPS II Regeneration Buffer (BioRad #153-6166

(7) 1M Tris, pH 9-11

(8) PBS

2. 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.

Wash column with 5-6 bed volumes of Binding Buffer. Collect 1 ml fractions until base line is achieved.

Elute Ab with 5 bed volumes of Elution Buffer. Collect 1 ml fractions into 50 ml 1M Tris, until base line is achieved.

Take 50 ml aliquots of each fraction for ELISA.

Pool fractions containing Ab. Dialyze against 2 x 4L PBS, overnight, 2-8℃.

Re-equilibrate column with Binding Buffer. Store column at 2-8℃.

Determine protein concentration of Ab, aliquot, and store at -20℃.

3. Note

Extinction Coefficient for IgG = 1.4 and for IgM = 1.2