Hashida Shin-Nosuke, Kawai-Yamada Maki
Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, Abiko, Japan.
Graduate School of Science and Engineering, Saitama University, Saitama, Japan.
Bio Protoc. 2019 Mar 5;9(5):e3183. doi: 10.21769/BioProtoc.3183.
Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple three-step method for detection of disulfide bonds in proteins. After irreversible blocking of protein thiols, disulfide bonds are reduced, followed by the detection of thiols. The approach presented here provides an economical procedure that can be used to obtain a global overview of the thiol-disulfide status of proteins in plants. This method allows the detection of modifications in samples on a gel and can be used for semi-quantitative analysis.
硫醇-二硫键交换是一种关键的翻译后修饰，决定着蛋白质内部和蛋白质间结构的折叠过程。硫醇可以通过比色试剂检测，比色试剂会被游离硫醇化学计量地还原，也可以通过荧光加合物检测，荧光加合物只有在硫酯形成后才会发出荧光。我们采用了一种简单的三步法来检测蛋白质中的二硫键。在蛋白质硫醇被不可逆封闭后，二硫键被还原，随后检测硫醇。本文介绍的方法提供了一种经济的程序，可用于全面了解植物中蛋白质的硫醇-二硫键状态。该方法能够检测凝胶上样品中的修饰情况，可用于半定量分析。
