Protein G Purification of Antibodies
Protein G Purification of AntibodiesSource: Contributed by Nanci DonackiReagent
| Protein G Purification of Antibodies |
| Source: Contributed by Nanci Donacki |
| Reagent and Materials Hi-Trap Protein G Column (Pharmacia Biotech #17-0404-01) 20 mM Sodium Phosphate Buffer, pH 7.0 1.084 g NaH2 PO4 , anhydrous 3.273 g Na2 HPO4 . 7H2 O q.s. to 1 liter with di-H2 O 0.1M Glycine, pH 2.8 3.75 g Glycine 1.4 ml HCl, concentrated q.s. to 1 liter with di-H2 O 1M Tris 141.1 g Tris base q.s. to 1 liter with di-H2 O. 20% Ethanol Procedure Prepare the collection tubes by adding 0.1 ml of 1M Tris per ml of each fraction to be collected. Centrifuge or filter the sample to remove any particulates. Adjust to pH 7.0. Wash the column with 5 bed volumes of 20 mM Sodium Phosphate Buffer, pH 7.0. Apply the sample onto the column. Was with 5 bed volumes of 20 mM Phosphate Buffer, pH 7.0 Elute the antibody with 1-3 bed volumes of 0.1M Glycine, collecting fractions into tubes containing 1M Tris. Regenerate column with 2-% Ethanol and store at 2-8o C. Pool fractions containing antibody and dialyze against 3 changes of PBS, at least 100 times the sample volume. Determine the protein concentration of the antibody. Concentrate if necessary. Store aliquots at -20o C. |
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