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        Protein A Purification of Antibody

        互联网

        4048

        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

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