Kikuchi Shingo, Bédard Jocelyn, Nakai Masato
Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
Methods Mol Biol. 2011;775:3-17. doi: 10.1007/978-1-61779-237-3_1.
Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful method for separating protein complexes from biological membranes under native conditions. BN-PAGE provides much higher resolution than gel filtration or sucrose density gradient centrifugation, and it can be used to estimate the molecular mass of protein complexes. First, membrane protein complexes need to be solubilized with a mild nonionic detergent such as digitonin or dodecyl maltoside. Coomassie brilliant blue G-250, a negatively charged dye that binds to the surface of the solubilized complexes, is then added so these can be resolved according to their size by non-denaturing (native) electrophoresis. BN-PAGE can be combined with a second dimension SDS-PAGE step (two-dimensional (2D)-BN/SDS-PAGE), so that the subunits making up these complexes are also separated according to their size. Here, we present our 2D-BN/SDS-PAGE method, and subsequent immunoblotting method, for the detection of relatively low-abundance proteins from plant chloroplasts.
蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）是一种在天然条件下从生物膜中分离蛋白质复合物的强大方法。与凝胶过滤或蔗糖密度梯度离心相比，BN-PAGE具有更高的分辨率，并且可用于估计蛋白质复合物的分子量。首先，膜蛋白复合物需要用温和的非离子去污剂（如洋地黄皂苷或十二烷基麦芽糖苷）进行增溶。然后加入考马斯亮蓝G-250，一种带负电荷的染料，它与增溶复合物的表面结合，这样就可以通过非变性（天然）电泳根据其大小进行分离。BN-PAGE可以与二维SDS-PAGE步骤（二维（2D）-BN/SDS-PAGE）相结合，从而使构成这些复合物的亚基也能根据其大小进行分离。在此，我们展示用于检测植物叶绿体中相对低丰度蛋白质的二维BN/SDS-PAGE方法及后续免疫印迹方法。
