Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.
Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.
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DOI：
10.1126/stke.3452006pl4
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发表时间：
2006-07-25
期刊：
影响因子：
--
通讯作者：
Schamel, Wolfgang W A
中科院分区：
文献类型：
--
作者：
Swamy, Mahima;Siegers, Gabrielle M;Schamel, Wolfgang W A
多蛋白复合物（MPC）在细胞信号转导中起着至关重要的作用。可以区分两种MPC：（i）组成型、丰富的MPC-例如，多亚基受体或转录因子;和（ii）信号诱导的、瞬时的、低拷贝数的MPC-例如，在含有Src-同源2（SH 2）结构域的蛋白质与酪氨酸磷酸化蛋白质结合时形成的复合物。蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）是一种比凝胶过滤或蔗糖密度超离心具有更高分辨率的分离方法，可用于分析10 kD至10 MD的丰富、稳定的MPC。与免疫沉淀和双杂交方法相反，它允许确定MPC的大小、相对丰度和亚基组成。此外，它还显示了有多少不同的复合物存在共享一个共同的亚基，是否存在单个亚基的游离单体形式，以及这些参数是否在细胞刺激后发生变化。在这里，我们给出了一个详细的协议，从总细胞裂解液或预纯化的MPC的分离通过一维BN-PAGE或通过二维BN-PAGE和SDS-PAGE。
Multiprotein complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCs--for example, multisubunit receptors or transcription factors; and (ii) signal-induced, transient, low copy number MPCs--for example, complexes that form upon binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a separation method with a higher resolution than gel filtration or sucrose density ultracentrifugation that can be used to analyze abundant, stable MPCs from 10 kD to 10 MD. In contrast to immunoprecipitation and two-hybrid approaches, it allows the determination of the size, the relative abundance, and the subunit composition of an MPC. In addition, it shows how many different complexes exist that share a common subunit, whether free monomeric forms of individual subunits exist, and whether these parameters change upon cell stimulation. Here, we give a detailed protocol for the separation of MPCs from total cellular lysates or of prepurified MPCs by one-dimensional BN-PAGE or by two-dimensional BN-PAGE and SDS-PAGE.