Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
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DOI：
10.3791/2164
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发表时间：
2011-02-01
影响因子：
1.2
通讯作者：
Blumenthal, Britta
中科院分区：
文献类型：
--
作者：
Fiala, Gina J.;Schamel, Wolfgang W. A.;Blumenthal, Britta
多蛋白复合体(MPC)在细胞信号传递中起着至关重要的作用，因为大多数蛋白质都可以在与其他蛋白质的功能或调节复合体中找到(Sali，Glaeser等人)。2003年)。因此，蛋白质-蛋白质相互作用网络的研究需要对MPC进行详细的表征，以获得对蛋白质功能和调控的综合理解。为了鉴定和分析，必须在本地条件下分离MPC。在这段视频中，我们描述了用蓝色天然聚丙烯酰胺凝胶电泳法(BN-PAGE)分析MPC。BN-PAGE是一种允许以比凝胶过滤或蔗糖密度超速离心法提供的更高分辨率来分离天然构象中的MPC的技术，因此对于确定MPC的大小、组成和相对丰度是有用的(Schagger和von Jagow 1991)；(Schagger，Cramer等人)。(1994年)。通过这种方法，蛋白质在聚丙烯酰胺基质中根据其流体力学大小和形状进行分离。在这里，我们演示了总细胞裂解物的MPC的分析，指出裂解液透析是使BN-PAGE适用于这些生物样品的关键步骤。结合一维BN-PAGE和二维SDSPAGE，我们发现BN-PAGE分离的MPC可以通过SDSPAGE进一步细分为各自的组分。凝胶分离时MPC组分的可视化是通过标准免疫印迹进行的。作为应用BN-PAGE进行MPC分析的一个例子，我们选择了真核生物19S、20S和26S蛋白酶体。
Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schagger and von Jagow 1991); (Schagger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.