Braakman Ineke, Lamriben Lydia, van Zadelhoff Guus, Hebert Daniel N
Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts.
Curr Protoc Protein Sci. 2017 Nov 1;90:14.1.1-14.1.21. doi: 10.1002/cpps.43.
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.
本单元提供了用于检测完整细胞培养物以及含有分离的微粒体或半透化细胞的体外翻译系统中二硫键形成的实验方案。首先，将新合成的目标蛋白在短时间脉冲中用放射性氨基酸进行生物合成标记。然后用未标记的氨基酸进行追踪。在追踪过程中的不同时间点，收集样品，用去污剂裂解膜，并按照所述方法通过免疫沉淀分离蛋白质。提供了一个辅助实验方案，用于通过SDS-PAGE在有或没有预先还原的情况下分析免疫沉淀物中的二硫键。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。还包括一个额外的辅助实验方案，该方案使用聚乙二醇马来酰亚胺修饰游离巯基，并通过SDS-PAGE追踪二硫键的形成。© 2017约翰威立国际出版公司。
