Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling

This chapter contains sections titled: Introduction Methods available Plasma membrane domains Analysis of membrane compartments in the endoplasmic reticulum–Golgi–plasma membrane pathway Separation of membrane vesicles from cytosolic proteins Endocytosis Protocols 5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients Protocols 5.2 Isolation of rat liver sinusoidal domain using antibody‐bound beads Protocols 5.3 Fractionation of apical and basolateral domains from Caco‐2 cells in a sucrose gradient Protocols 5.4 Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient Protocols 5.5 Isolation of lipid rafts Protocols 5.6 Isolation of caveolae Protocols 5.7 Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose–D 2 O density gradients Protocols 5.8 Analysis of Golgi, ER, ERGIC and other membrane compartments from cultured cells using continuous iodixanol density gradients Protocols 5.9 Analysis of Golgi, ER, TGN and other membrane compartments in sedimentation velocity iodixanol density gradients (continuous or discontinuous) Protocols 5.10 SDS–PAGE of membrane proteins Protocols 5.11 Semi‐dry blotting Protocols 5.12 Detection of blotted proteins by enhanced chemiluminescence (ECL) Protocols 5.13 Separation of membranes and cytosolic fractions from (a) mammalian cells and (b) bacteria Protocols 5.14 Analysis of early and recycling endosomes in preformed iodixanol gradients; endocytosis of transferrin in transfected MDCK cells Protocols 5.15 Analysis of clathrin‐coated vesicle processing in self‐generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver Protocols 5.16 Polysucrose–Nycodenz ® gradients for the analysis of dense endosome–lysosome events in mammalian liver References