Santoni Véronique
Biochimie et Physiologie Moléculaire des Plantes, INRA, Montpellier, France.
Methods Mol Biol. 2007;355:93-109. doi: 10.1385/1-59745-227-0:93.
The plasma membrane (PM) exists as the interface between the cytosol and the environment in all living cells and is one of the most complex and differentiated membrane. The identification and characterization of membrane proteins (either extrinsic or intrinsic) is a crucial challenge since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism. Methods to isolate PM fractions vary according to organisms, tissues, and cell type. This chapter emphasizes isolation, from the model plant Arabidopsis thaliana, of PM fractions from a microsomal membrane fraction by two-phase partitioning, a methodology that utilizes the different surface properties of membranes. PM proteins that do not span the lipid bilayer are generally well recovered after 2D gel electrophoresis. By contrast, the recovery of transmembrane proteins requires first the depletion of the PM fraction from soluble proteins, being either cytosolic contaminants or functionally associated proteins, and second, to the use of specific solubilization procedures. This chapter presents protocols to strip PM based on alkaline treatment of membranes and to solubilize hydrophobic proteins to increase their recovery on 2D gels. Aquaporins that are highly hydrophobic proteins are used to probe the relevance of the procedures.
质膜（PM）作为所有活细胞中胞质溶胶与外界环境的界面而存在，是最为复杂且分化程度最高的膜结构之一。膜蛋白（外在蛋白或内在蛋白）的鉴定与表征是一项关键挑战，因为这些蛋白中有许多参与细胞的基本功能，如细胞信号传导、渗透调节、营养和代谢。分离质膜组分的方法因生物、组织和细胞类型而异。本章重点介绍通过两相分配法从模式植物拟南芥的微粒体膜组分中分离质膜组分，这是一种利用膜表面不同性质的方法。不跨越脂质双层的质膜蛋白通常在二维凝胶电泳后能得到较好的回收。相比之下，跨膜蛋白的回收首先需要去除质膜组分中的可溶性蛋白，这些可溶性蛋白可能是胞质污染物或功能相关蛋白，其次需要使用特定的增溶程序。本章介绍了基于膜的碱性处理去除质膜以及增溶疏水蛋白以提高其在二维凝胶上回收率的方法。水通道蛋白是高度疏水的蛋白，被用于探究这些方法的相关性。
