用于研究细胞内运输的受体酪氨酸激酶的细胞表面生物素化

Cell surface biotinylation of receptor tyrosine kinases to investigate intracellular trafficking.

作者信息

Crupi Mathieu J F, Richardson Douglas S, Mulligan Lois M

机构信息

Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Botterell Hall Rm A315, Kingston, ON, Canada, K7L 3N6.

出版信息

Methods Mol Biol. 2015;1233:91-102. doi: 10.1007/978-1-4939-1789-1_9.

DOI:10.1007/978-1-4939-1789-1_9

PMID:25319892

Abstract

Cell surface biotinylation is a biochemical approach to covalently bind membrane-impermeable biotin to the extracellular domain of membrane proteins, such as receptor tyrosine kinases (RTKs). Subsequent to ligand incubation periods, activated biotinylated receptors may internalize from the cell surface into early endosomes and then travel through intracellular compartments to either recycle back to the membrane or degrade in lysosomes. The biotin-labeled proteins may be detected through affinity purification with streptavidin agarose resins. This chapter describes methods for cell surface biotinylation to assess RTK trafficking steps.

摘要

细胞表面生物素化是一种生化方法，用于将膜不可渗透的生物素共价结合到膜蛋白的细胞外结构域，如受体酪氨酸激酶（RTK）。在配体孵育期之后，活化的生物素化受体可能从细胞表面内化到早期内体中，然后通过细胞内区室运输，要么循环回到细胞膜，要么在溶酶体中降解。生物素标记的蛋白质可以通过用链霉亲和素琼脂糖树脂进行亲和纯化来检测。本章描述了用于评估RTK运输步骤的细胞表面生物素化方法。