Protein A Purification of Antibody
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Reagents
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Affi-gel Protein-A Agarose (BioRad #153-6153)
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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
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1M Tris, pH 9-11
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PBS
Procedure
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Dilute Ab 1:5 with Binding Buffer. Filter through a 0.22 m filter.
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Equilibrate column with at least 2 bed volumes of Binding Buffer
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Apply Sample.
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Wash column with 5-6 bed volumes of Binding Buffer. Collect 1 ml fractions until base line is achieved.
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Elute Ab with 5 bed volumes of Elution Buffer. Collect 1 ml fractions into 50 m l 1M Tris, until base line is achieved.
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Take 50 m l aliquots of each fraction for ELISA.
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Pool fractions containing Ab. Dialyze against 2 x 4L PBS, overnight, 2-8o C.
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Re-equilibrate column with Binding Buffer. Store column at 2-8o C.
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Determine protein concentration of Ab, aliquot, and store at -20o C.
Note
Extinction Coefficient for IgG = 1.4 and for IgM = 1.2