Yoshihara Y, Kuroda M
Department of Biology, Faculty of Sciences, Shimane University.
J Biochem. 1995 Feb;117(2):443-6. doi: 10.1093/jb/117.2.443.
This paper describes a simple and efficient method for preparing affinity columns. We used protein separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as a ligand. Protein bands detected in a polyacrylamide gel were electrophoretically transferred to CNBr-activated Sepharose using a buffer containing Nonidet P-40. The amount of ligand protein coupled to activated Sepharose by our method was almost comparable to that obtained by conventional coupling procedure with a native ligand protein. Using affinity columns prepared by this method, we have successfully purified anti-alpha-actinin antibody and antibodies highly specific to the rod domain of alpha-actinin from the antiserum. This new method should be useful for separating a specific antibody from an antiserum that has been raised against multiple antigens. In addition, the use of the SDS-gel-fractionated band facilitates the coupling of proteins that have low solubility under the coupling conditions.
本文描述了一种制备亲和柱的简单有效方法。我们使用经十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳分离的蛋白质作为配体。在聚丙烯酰胺凝胶中检测到的蛋白带通过含有聚乙二醇辛基苯基醚的缓冲液电泳转移至溴化氰活化的琼脂糖凝胶上。通过我们的方法与活化琼脂糖凝胶偶联的配体蛋白量几乎与使用天然配体蛋白的传统偶联方法所获得的量相当。使用以此方法制备的亲和柱，我们已成功从抗血清中纯化出抗α-辅肌动蛋白抗体以及对α-辅肌动蛋白杆状结构域具有高度特异性的抗体。这种新方法对于从针对多种抗原产生的抗血清中分离特异性抗体应是有用的。此外，使用SDS凝胶分级分离带便于偶联在偶联条件下溶解度低的蛋白质。
