Protein A Purification of Antibody

Reagents

  Affi-gel Protein-A Agarose (BioRad #153-6153)   MAPS II Binding Buffer (BioRad # 153-6161) 0.314 g/ml diH2 O MAPS II Elution Buffer (BioRad #153-6162) 0.023 g/ml diH2 O MAPS II Regeneration Buffer (BioRad #153-6166 1M Tris, pH 9-11 PBS

  Procedure

Dilute Ab 1:5 with Binding Buffer.  Filter through a 0.22 m filter. Equilibrate column with at least 2 bed volumes of Binding Buffer 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 m l 1M Tris, until base line is achieved. Take 50 m l aliquots of each fraction for ELISA. Pool fractions containing Ab.  Dialyze against 2 x 4L PBS, overnight, 2-8o C. Re-equilibrate column with Binding Buffer.  Store column at 2-8o C.   Determine protein concentration of Ab, aliquot, and store at -20o C.

Note

 Extinction Coefficient for IgG = 1.4 and for IgM = 1.2