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

• Published in: Journal of biochemical and biophysical methods 2007-04, Vol.70 (3), p.493-498
• Main Authors: Arakawa, Tsutomu, Tsumoto, Kouhei, Ejima, Daisuke, Kita, Yoshiko, Yonezawa, Yasushi, Tokunaga, Masao
• Format: Article
• Language: English
• Subjects: Ammonium Sulfate; Animals; Blue-Sepharose; Cattle; Chromatography, Affinity - methods; Chromatography, Ion Exchange - methods; Dye-affinity; Protein Binding; Proteins - isolation & purification; Q-Sepharose; Salting-out; Sepharose - analogs & derivatives; Serum Albumin, Bovine - isolation & purification
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/j.jbbm.2006.12.001

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.