Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography
Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography
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DOI：
10.1016/j.jbbm.2006.12.001
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发表时间：
2007-04-10
影响因子：
--
通讯作者：
Tokunaga, Masao
中科院分区：
文献类型：
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作者：
Arakawa, Tsutomu;Tsumoto, Kouhei;Tokunaga, Masao
一般来说，蛋白质在低盐浓度下结合到亲和柱或离子交换柱上，通过提高盐浓度、改变溶剂pH值或添加竞争配体来洗脱结合的蛋白质。Blue-Sepharose通常用于去除样品中的牛血清白蛋白（BSA），但当我们将BSA应用于Blue-Sepharose，在20 mM磷酸盐，pH 7.0中，50%-60%的蛋白流过柱；然而，通过在柱平衡缓冲液和样品中加入2 M硫酸铵（AS），可以实现BSA的完全结合。通过降低AS浓度或加入1m NaCl或精氨酸洗脱结合蛋白。高浓度的AS在pH 7.0时甚至导致BSA与离子交换柱Q-Sepharose结合。因此，虽然适度的盐浓度可以从蓝- sepharose或离子交换柱中洗脱蛋白质，但在极端的盐析条件下，蛋白质可以与这些柱结合。在atp亲和柱上观察到AS对蛋白质的类似增强结合。(c) 2006 Elsevier B.V.版权所有
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 I 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. (c) 2006 Elsevier B.V. All rights reserved.