Kinoshita-Kikuta Emiko, Kinoshita Eiji, Koike Tohru
Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.
Methods Mol Biol. 2015;1295:323-54. doi: 10.1007/978-1-4939-2550-6_24.
In this chapter, we describe a standard protocol for phosphate-affinity SDS-PAGE that uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system (Zn(2+-)Phos-tag SDS-PAGE) to detect shifts in the mobilities of phosphoproteins. A previous protocol for affinity electrophoresis that uses polyacrylamide-bound Mn(2+)-Phos-tag and Laemmli's buffer system under conditions of alkaline pH has limitations in separating certain phosphoproteins. The current protocol provides major improvements in separation and detection of various phosphorylated protein species. We here introduce two neutral-pH gel systems buffered with Bis-Tris-HCl and Tris-AcOH, respectively, for Zn(2+)-Phos-tag SDS-PAGE, and we also discuss their characteristics on the basis of comparative studies on phosphorylation profiling of proteins with a wide range of molecular masses. Each analytical procedure, from the beginning of gel preparation to the end of electrophoresis, requires 2.5-5 h with either buffer system.
在本章中，我们描述了一种用于磷酸亲和SDS-PAGE的标准方案，该方案使用磷酸结合分子Phos-tag的二锌（II）配合物与中性pH凝胶系统（Zn(2+)-Phos-tag SDS-PAGE）相结合，以检测磷蛋白迁移率的变化。先前一种使用聚丙烯酰胺结合的Mn(2+)-Phos-tag和Laemmli缓冲系统在碱性pH条件下进行亲和电泳的方案，在分离某些磷蛋白方面存在局限性。当前方案在各种磷酸化蛋白种类的分离和检测方面有了重大改进。我们在此分别介绍两种用Bis-Tris-HCl和Tris-AcOH缓冲的中性pH凝胶系统用于Zn(2+)-Phos-tag SDS-PAGE，并且我们还基于对具有广泛分子量的蛋白质磷酸化谱的比较研究讨论了它们的特性。从凝胶制备开始到电泳结束，使用任何一种缓冲系统，每个分析过程都需要2.5 - 5小时。
