Eichacker Lutz Andreas, Weber Gerhard, Sukop-Köppel Ute, Wildgruber Robert
Center of Organelle Research (CORE), University of Stavanger, Richard Johnsensgate 4, 4036, Stavanger, Norway.
Methods Mol Biol. 2015;1295:415-25. doi: 10.1007/978-1-4939-2550-6_29.
This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate membrane protein complexes for proteome analysis. FFE is a highly versatile technology applied in the field of protein analysis. It is superior to native PAGE due to its fast continuous processing of sample at high resolution. Additionally, the dynamic separation range from ions, peptides, to proteins, protein complexes, up to organelles, and whole cells makes it the method of choice in the analysis of proteins. FFE is carried out in an aqueous medium without inducing any solid matrix, such as acrylamide, so that it simplifies the analysis of protein complexes for the downstream analysis. Here, we describe the novel zone electrophoresis interval method (IZE-FFE) for separation of protein complexes from the thylakoid membrane of Arabidopsis thaliana by charge only. Protein complexes isolated by IZE FFE were characterized according to molecular weight by Blue Native PAGE and were proteins stained with coomassie.
本章介绍了自由流动电泳（FFE）技术以及用于分离膜蛋白复合物以进行蛋白质组分析的实验方案。FFE是一种应用于蛋白质分析领域的高度通用的技术。由于它能以高分辨率快速连续处理样品，因此优于天然聚丙烯酰胺凝胶电泳（native PAGE）。此外，其从离子、肽到蛋白质、蛋白质复合物，直至细胞器和全细胞的动态分离范围，使其成为蛋白质分析中的首选方法。FFE在水性介质中进行，不会诱导任何固体基质，如丙烯酰胺，从而简化了用于下游分析的蛋白质复合物的分析。在此，我们描述了一种新型的区带电泳间隔法（IZE-FFE），该方法仅根据电荷从拟南芥类囊体膜中分离蛋白质复合物。通过IZE-FFE分离的蛋白质复合物通过蓝色天然聚丙烯酰胺凝胶电泳（Blue Native PAGE）根据分子量进行表征，并用考马斯亮蓝对蛋白质进行染色。
