Weiland Florian, Zammit Carla M, Reith Frank, Hoffmann Peter
Adelaide Proteomics Centre, University of Adelaide, Adelaide, Australia.
Electrophoresis. 2014 Jul;35(12-13):1893-902. doi: 10.1002/elps.201400060. Epub 2014 Apr 16.
Blue native PAGE (BN-PAGE) is a powerful method to separate protein complexes while preserving their native state. However, the resolution of the method is limited as complexes with similar molecular masses cannot be resolved. Here we describe native 2DE using immobilized pH-gradients in combination with BN-PAGE to resolve protein complexes by their pI and molecular mass. This method enables electrophoretic separation of proteins between pI 3 and 10 and can resolve molecular masses up to 1.2 MDa. Visualized gel spots at large molecular weight were identified using MS to confirm potential protein complexes. Several protein complexes could be identified, most prominent GroEL in complex with GroES, parts of the ribosomal machinery and membrane transport system. In summary, this method enables easy high-resolution electrophoretic separation of protein complexes.
蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）是一种在保持蛋白质复合物天然状态的同时分离它们的强大方法。然而，该方法的分辨率有限，因为具有相似分子量的复合物无法区分。在此，我们描述了使用固定化pH梯度结合BN-PAGE的天然双向电泳，通过蛋白质等电点（pI）和分子量来解析蛋白质复合物。这种方法能够对pI在3至10之间的蛋白质进行电泳分离，并且可以分辨高达1.2 MDa的分子量。利用质谱对高分子量处可视化的凝胶斑点进行鉴定，以确认潜在的蛋白质复合物。鉴定出了几种蛋白质复合物，最显著的是与GroES形成复合物的GroEL、核糖体机制的部分成分以及膜转运系统。总之，这种方法能够轻松地对蛋白质复合物进行高分辨率电泳分离。
