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Protein A Purification of Antibody

Author: Nanci Donacki
Source: Contributed by Nanci Donacki
Date Added: Tue May 14 2002
Date Modified: Thu Apr 29 2004
Abstract: Protein A Purification of Antibody - MAPS II System

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 0.22æ 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 æl 1M Tris, until base line is achieved.
  6. Take 50 æl aliquots of each fraction for ELISA.
  7. Pool fractions containing Ab.  Dialyze against 2 x 4L PBS, overnight, 2-8oC.
  8. Re-equilibrate column with Binding Buffer.  Store column at 2-8oC.
  9.  Determine protein concentration of Ab, aliquot, and store at -20oC.

Note

 Extinction Coefficient for IgG = 1.4 and for IgM = 1.2

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