Yoshida Keisuke, Hisabori Toru
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan.
Bio Protoc. 2019 Jun 5;9(11):e3250. doi: 10.21769/BioProtoc.3250.
Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state . Here we present a simple procedure to determine protein redox state in a model plant . Our method consists of three key steps: (i) redox fixation by rapidly freezing plant tissues in the liquid nitrogen, (ii) labeling of thiol groups with the maleimide reagent, and (iii) protein detection by Western blotting. The redox state of a specific or given protein can be discriminated by the mobility change on SDS-PAGE with high sensitivity. This method provides a novel strategy to dissect the working dynamics of the redox-regulatory system in plants.
基于硫醇的氧化还原调节是一种翻译后蛋白质修饰，在许多生物学方面发挥着关键作用。为了解其调节功能，我们需要一种直接评估蛋白质氧化还原状态的方法。在此，我们提出了一种在模式植物中确定蛋白质氧化还原状态的简单程序。我们的方法包括三个关键步骤：（i）通过在液氮中快速冷冻植物组织进行氧化还原固定，（ii）用马来酰亚胺试剂标记硫醇基团，以及（iii）通过蛋白质印迹法进行蛋白质检测。特定或给定蛋白质的氧化还原状态可以通过SDS-PAGE上的迁移率变化以高灵敏度进行区分。该方法为剖析植物氧化还原调节系统的工作动态提供了一种新策略。
