Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Anal Chem. 2010 Jan 15;82(2):755-61. doi: 10.1021/ac902290q.
A method for the affinity capture of specific proteins from a complex mixture using a polyacrylamide gel technique is described. The approach is based on the orthogonal electro-transfer of proteins separated by ordinary polyacrylamide gel electrophoresis (PAGE) to a ligand-coupled polyacrylamide gel (Li-PAG), which is placed under the PAGE gel. Upon electro-transfer, the proteins orthogonally migrate from the PAGE into the Li-PAG, based on the net charge. During migration to the Li-PAG, proteins that specifically interact with a ligand can be transiently trapped in the Li-PAG, while those that do not interact with a ligand pass through it. This method permits the separation of the proteins that can specifically interact with a ligand, even when present in a complex mixture. The method is demonstrated by applying it to the one-step isolation of a trypsin inhibitor from a crude extract of soybean flour.
本文描述了一种使用聚丙烯酰胺凝胶技术从复杂混合物中亲和捕获特定蛋白质的方法。该方法基于通过普通聚丙烯酰胺凝胶电泳（PAGE）分离的蛋白质的正交电转移到与配体偶联的聚丙烯酰胺凝胶（Li-PAG），该凝胶放置在 PAGE 凝胶下方。在电转移过程中，基于净电荷，蛋白质从 PAGE 正交迁移到 Li-PAG。在迁移到 Li-PAG 的过程中，与配体特异性相互作用的蛋白质可以暂时滞留在 Li-PAG 中，而那些不与配体相互作用的蛋白质则穿过它。即使在复杂混合物中，该方法也允许分离可以与配体特异性相互作用的蛋白质。该方法通过将其应用于从大豆粉粗提物中一步分离胰蛋白酶抑制剂来进行演示。
