LaMontagne Erica D, Collins Carina A, Peck Scott C, Heese Antje
University of Missouri-Columbia, Division of Biochemistry, Interdisciplinary Plant Group (IPG), Columbia, Missouri.
Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri.
Curr Protoc Plant Biol. 2016 May;1(1):217-234. doi: 10.1002/cppb.20020.
Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms, mediating transport of molecules, and facilitating trafficking of cargo between cellular compartments. Because membrane functionality is largely defined by protein composition, exploring the roles of membrane proteins is of interest to many researchers. This article focuses on the subcellular fractionation of microsomes, which are membrane-derived vesicles formed during cell lysis. In plants, microsomes mainly consist of the plasma membrane and membranes derived from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and tonoplast. The article describes the different steps involved in enriching for and solubilizing microsomal membrane proteins from Arabidopsis thaliana seedlings and cultured cells by differential centrifugation. Solubilized microsomal proteins can be used for subsequent immunoblot analysis, co-immunoprecipitation, or proteomic studies. © 2016 by John Wiley & Sons, Inc.
细胞膜界定了细胞器与细胞质溶胶或细胞外环境之间的边界，从而在亚细胞区室之间实现功能分隔。此外，膜有助于多种细胞功能，包括作为信号平台、介导分子运输以及促进细胞区室之间的货物运输。由于膜的功能很大程度上由蛋白质组成决定，因此探索膜蛋白的作用受到许多研究人员的关注。本文聚焦于微粒体的亚细胞分级分离，微粒体是细胞裂解过程中形成的膜衍生囊泡。在植物中，微粒体主要由质膜以及源自内质网、高尔基体、反式高尔基体网络和液泡膜的膜组成。本文描述了通过差速离心从拟南芥幼苗和培养细胞中富集并溶解微粒体膜蛋白所涉及的不同步骤。溶解的微粒体蛋白可用于后续的免疫印迹分析、免疫共沉淀或蛋白质组学研究。© 2016 约翰威立父子公司版权所有
