Nakatogawa Hitoshi, Ohsumi Yoshinori
Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan.
Methods Mol Biol. 2012;832:519-29. doi: 10.1007/978-1-61779-474-2_37.
Autophagy is a bulk degradation system highly conserved among eukaryotic cells and plays crucial roles in a wide range of physiological and pathological situations. Remarkably, this process involves two ubiquitin-like (Ubl) conjugation systems. Here, we describe two sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques to analyze these systems: one that allows separation of the Ubl protein Atg8 conjugated to the lipid phosphatidylethanolamine from its unlipidated form, and the other by which otherwise labile thioester intermediates between Atg8 and either the E1 enzyme Atg7 or the E2 enzyme Atg3 are stably preserved during electrophoresis, and thus easily detected by following protein visualization. Especially, the latter technique is also ubiquitously applicable for studies on conjugation reactions of ubiquitin (Ub) and other Ubl proteins.
自噬是真核细胞中高度保守的一种大量降解系统，在多种生理和病理情况下发挥关键作用。值得注意的是，这一过程涉及两个类泛素（Ubl）缀合系统。在此，我们描述两种十二烷基硫酸钠-聚丙烯酰胺凝胶电泳技术来分析这些系统：一种可将与脂质磷脂酰乙醇胺缀合的Ubl蛋白Atg8与其未脂质化形式分离，另一种可使Atg8与E1酶Atg7或E2酶Atg3之间原本不稳定的硫酯中间体在电泳过程中得到稳定保存，从而通过蛋白质可视化追踪轻易检测到。特别地，后一种技术也普遍适用于泛素（Ub）和其他Ubl蛋白缀合反应的研究。
