高尔基体网络中货物蛋白包装到囊泡过程的无细胞重建
Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the Golgi Network.
作者信息
Tang Xiao, Yang Feng, Guo Yusong
机构信息
Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China.
出版信息
Bio Protoc. 2020 Mar 5;10(5):e3537. doi: 10.21769/BioProtoc.3537.
Abstract
Protein sorting at the Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. The protocol describes how to perform the vesicle formation assay, how to isolate vesicles and how to detect cargo proteins in vesicles. This reconstitution assay can be used to quantitatively measure the efficiency of the packaging of a specific cargo protein into transport vesicles at the TGN under specific experimental conditions.
摘要
高尔基体网络（TGN）中的蛋白质分选在将新合成的蛋白质靶向其特定目的地方面发挥着重要作用。本提议的目的是利用大鼠肝细胞溶胶、半完整哺乳动物细胞和核苷酸，在TGN处将非高尔基体驻留货物蛋白重新包装到囊泡中。该方案描述了如何进行囊泡形成测定、如何分离囊泡以及如何检测囊泡中的货物蛋白。这种重组测定可用于在特定实验条件下定量测量特定货物蛋白在TGN处包装到运输囊泡中的效率。
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