Affinity Chromatography: Methods and Protocols by Meir Wilchek, Irwin Chaiken (auth.), Pascal Bailon, George

By Meir Wilchek, Irwin Chaiken (auth.), Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, Wolfgang Berthold (eds.)

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M. and Marchalonis, J. J. (1978) A rapid, novel method for the solidphase derivatization of IgG antibodies for immune-affinity chromatography. J. Immunol. Methods 127, 215–220. 10. , Newman, R. , Sutherland, D. , and Greaves, M. F. (1982) One step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Chem. 257, 10,766–10,769. 11. Sisson, T. H. and Castor, C. W. (1990) An improved method for immobilizing IgG antibodies on protein A-agarose. J. Immunol. Methods 127, 215–220.

Upon turning yellow, the reagent should be discarded. 4. 0 5. 0. 44 Podlaski and Stern 3. 1. Binding Purified IgG to Protein G Sepharose 1. 2, and wash three times with 10 vol in a 50-mL polypropylene tube. Use a benchtop centrifuge at approx 1500g for 1 min to separate the beads from the wash buffer. 2. Add the antibody to the washed Protein G Sepharose to obtain a density of 2 mg of IgG/mL of Protein G Sepharose. Incubate for 30 min at room temperature on a rotary shaker. 3. 4 (average for most IgGs).

RIL-2 1. Fluidize receptor–affinity beads as in HAT purification procedure. 2. Apply 10 L of unclarified crude rIL-2 extract, enough to saturate the affinity gel, onto the column in FB mode at the same flow rate. 3. Wash away any unadsorbed materials using 10 cv of PBS at 30 mL/min in FB mode. After washing, stop flow and allow gel to settle. 4. Lower the top piston to meet the settled gel bed (column mode). 5. 3, at 30 mL/min (see Note 18). Monitor the protein peak at 280 nm with the UV detector contained in the TRIO automated system, and collect the peak.

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