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Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

Arakawa T, Tsumoto K, Ejima D, Kita Y, Yonezawa Y, Tokunaga M

Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA. tarakawa2@aol.com

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

Published 12 March 2007 in J Biochem Biophys Methods, 70(3): 493-8.
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