Braakman I, Hebert D N
University of Amsterdam, Academic Medical Center, The Netherlands.
Curr Protoc Protein Sci. 2001 May;Chapter 14:Unit14.1. doi: 10.1002/0471140864.ps1401s03.
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. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein is then 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 unreduced and reduced samples is due to disulfide bonds in the unreduced protein.
本单元提供了在完整细胞培养物和含有分离微粒体的体外翻译系统中检测二硫键形成的实验方案。首先，用放射性氨基酸对新合成的目标蛋白质进行短时间脉冲生物合成标记。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。还提供了一个辅助实验方案，用于通过SDS-PAGE分析免疫沉淀产物中的二硫键，包括未还原和还原处理。未还原样品和还原样品在凝胶上观察到的迁移率差异是由于未还原蛋白质中的二硫键所致。
