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        Protein A Purification of Antibody

        互联网

        948
         

        Reagents

        1.   Affi-gel Protein-A Agarose (BioRad #153-6153)
        2.   MAPS II Binding Buffer (BioRad # 153-6161)
          0.314 g/ml diH2 O
        3. MAPS II Elution Buffer (BioRad #153-6162)
          0.023 g/ml diH2 O
        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 m l 1M Tris, until base line is achieved.
        6. Take 50 m l aliquots of each fraction for ELISA.
        7. Pool fractions containing Ab.  Dialyze against 2 x 4L PBS, overnight, 2-8o C.
        8. Re-equilibrate column with Binding Buffer.  Store column at 2-8o C.
        9.   Determine protein concentration of Ab, aliquot, and store at -20o C.

        Note

         Extinction Coefficient for IgG = 1.4 and for IgM = 1.2

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