Wu Yuliang, Li Qiang, Chen Xing-Zhen
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Nat Protoc. 2007;2(12):3278-84. doi: 10.1038/nprot.2007.459.
Far western blotting (WB) was derived from the standard WB method to detect protein-protein interactions in vitro. In Far WB, proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and probed, usually with purified bait protein(s). The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey protein together form a complex. Compared with other biochemical binding assays, Far WB allows prey proteins to be endogenously expressed without purification. Unlike most methods using cell lysates (e.g., co-immunoprecipitation (co-IP)) or living cells (e.g., fluorescent resonance energy transfer (FRET)), Far WB determines whether two proteins bind to each other directly. Furthermore, in cases where they bind to each other indirectly, Far WB allows the examination of candidate protein(s) that form a complex between them. Typically, 2-3 d are required to carry out the experiment.
远西印迹法（Far western blotting，Far WB）源自标准的蛋白质印迹法（WB），用于在体外检测蛋白质-蛋白质相互作用。在远西印迹法中，首先如标准蛋白质印迹法那样，通过十二烷基硫酸钠（SDS）或非变性聚丙烯酰胺凝胶电泳（PAGE）分离含有猎物蛋白的细胞裂解液中的蛋白质，并将其转移至膜上。然后使膜上的蛋白质变性并复性。接着封闭膜并进行检测，通常使用纯化的诱饵蛋白。如果诱饵蛋白和猎物蛋白共同形成复合物，那么在膜上猎物蛋白所在的位置就能检测到诱饵蛋白。与其他生化结合分析方法相比，远西印迹法允许猎物蛋白在未经纯化的情况下内源性表达。与大多数使用细胞裂解液的方法（如免疫共沉淀法（co-IP））或活细胞的方法（如荧光共振能量转移法（FRET））不同，远西印迹法可确定两种蛋白质是否直接相互结合。此外，在它们间接相互结合的情况下，远西印迹法能够检测在它们之间形成复合物的候选蛋白。通常，进行该实验需要2至3天。
