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{"title":"Analysis of Disulfide Bond Formation","authors":"Ineke Braakman, Lydia Lamriben, Guus van Zadelhoff, Daniel N. Hebert","doi":"10.1002/cpps.43","DOIUrl":null,"url":null,"abstract":"<p>In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.43","citationCount":"21","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Protein Science","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpps.43","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 21
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Abstract
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley & Sons, Inc.
二硫键形成的分析
在本单元中，提供了检测完整细胞培养和含有分离微粒体或半透性细胞的体外翻译系统中二硫键形成的方案。首先，新合成的感兴趣的蛋白质在短脉冲中用放射性氨基酸进行生物合成标记。然后标记的蛋白质被未标记的氨基酸追赶。在追捕过程中的不同时间，收集样品，用洗涤剂裂解膜，并通过免疫沉淀分离蛋白质，如前所述。提供了一种支持方案，用于分析免疫沉淀中的二硫键，SDS-PAGE有或没有事先还原。在未还原和还原样品的凝胶之间观察到的迁移率差异是由于未还原蛋白质中的二硫键。附加的支持协议包括使用peg -马来酰亚胺修饰游离硫醇，并通过SDS-PAGE遵循二硫化物键的形成。©2017 by John Wiley &儿子,Inc。
本文章由计算机程序翻译，如有差异，请以英文原文为准。
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