Gloeckner Christian Johannes, Boldt Karsten, Ueffing Marius
Helmholtz Zentrum München, Neuherberg, Germany.
Technical University of Munich, Munich, Germany.
Curr Protoc Protein Sci. 2009 Aug;Chapter 19:19.20.1-19.20.19. doi: 10.1002/0471140864.ps1920s57.
In recent years, several methods have been developed to analyze protein-protein interactions under native conditions. One of them, tandem affinity purification (TAP), combines two affinity-purification steps to allow isolation of high-purity protein complexes. This unit presents a methodological workflow based on an SF-TAP tag comprising a doublet Strep-tag II and a FLAG moiety optimized for rapid as well as efficient tandem affinity purification of native proteins and protein complexes in higher eukaryotic cells. Depending on the stringency of purification conditions, SF-TAP allows both the isolation of a single tagged-fusion protein of interest and purification of protein complexes under native conditions.
近年来，已开发出几种方法来分析天然条件下的蛋白质-蛋白质相互作用。其中之一是串联亲和纯化（TAP），它结合了两个亲和纯化步骤，以实现高纯度蛋白质复合物的分离。本单元介绍了一种基于SF-TAP标签的方法流程，该标签由双联体链霉亲和素标签II和一个FLAG部分组成，经过优化，可在高等真核细胞中快速高效地对天然蛋白质和蛋白质复合物进行串联亲和纯化。根据纯化条件的严格程度，SF-TAP既能分离出感兴趣的单一标记融合蛋白，也能在天然条件下纯化蛋白质复合物。
