Young Mark T
School of Biosciences, Cardiff University, Cardiff, UK.
Methods Mol Biol. 2020;2041:137-146. doi: 10.1007/978-1-4939-9717-6_9.
Covalent labeling of protein with biotin (biotinylation) is a versatile technique which enables the capture and analysis of labeled protein with streptavidin-coated beads. It is particularly useful for detecting and quantifying the cell-surface expression of membrane proteins and widely used to analyze protein trafficking and the effect of mutations (particularly those which render the protein nonfunctional) on cell-surface expression. Here I describe the procedure for biotinylation and capture of cell-surface rat P2X2 receptors expressed in mammalian cells, and outline the steps in data analysis required to measure the proportion of cell-surface expressed protein of single point mutants relative to a wild-type control.
用生物素对蛋白质进行共价标记（生物素化）是一种通用技术，它能够利用包被抗生物素蛋白的磁珠捕获和分析标记的蛋白质。该技术对于检测和定量膜蛋白的细胞表面表达特别有用，并且广泛用于分析蛋白质运输以及突变（特别是那些使蛋白质失去功能的突变）对细胞表面表达的影响。在此，我描述了在哺乳动物细胞中表达的细胞表面大鼠P2X2受体的生物素化和捕获过程，并概述了测量单点突变体相对于野生型对照的细胞表面表达蛋白比例所需的数据分析步骤。
