Protein G Purification of Antibodies

1. Reagent and Materials

(1) Hi-Trap Protein G Column (Pharmacia Biotech #17-0404-01)

(2) 20 mM Sodium Phosphate Buffer, pH 7.0

1.084 g NaH₂PO₄, anhydrous

3.273 g Na₂HPO₄.7H2O

q.s. to 1 liter with di-H₂O

(3) 0.1M Glycine, pH 2.8

3.75 g Glycine

1.4 ml HCl, concentrated

q.s. to 1 liter with di-H₂O

(4) 1M Tris

141.1 g Tris base

q.s. to 1 liter with di-H₂O.

(5) 20% Ethanol

2. Procedure

(1) Prepare the collection tubes by adding 0.1 ml of 1M Tris per ml of each fraction to be collected.

(2) Centrifuge or filter the sample to remove any particulates. Adjust to pH 7.0.

(3) Wash the column with 5 bed volumes of 20 mM Sodium Phosphate Buffer, pH 7.0.

(4) Apply the sample onto the column.

(5) Wash with 5 bed volumes of 20 mM Phosphate Buffer, pH 7.0

(6) Elute the antibody with 1-3 bed volumes of 0.1M Glycine, collecting fractions into tubes ntaining 1M Tris.

(7) Regenerate column with 2-% Ethanol and store at 2-8℃.

(8) Pool fractions containing antibody and dialyze against 3 changes of PBS, at least 100 times the sample volume.

(9) Determine the protein concentration of the antibody. Concentrate if necessary. Store aliquots at -20℃.