Membrane analysis

1. General membrane composition and structure -- 2. The membranes of prokaryotes and eukaryotes -- 3. Preparation of subcellular membranes -- 4. Molecular composition -- 5. Investigating the topology of membrane proteins -- 6. Investigation of membrane lipids -- 7. Protein targetting and membrane biogenesis -- 8. Endocytosis and vesicular trafficking. Cover......Page 1 Half Title......Page 2 Series Page......Page 3 Title Page......Page 4 Copyright Page......Page 5 Table of Contents......Page 6 Abbreviations......Page 12 Preface......Page 14 General lipid composition......Page 16 Phospholipids......Page 19 Isoprenoids......Page 23 Lipid architecture......Page 24 Proteins......Page 25 Peripheral proteins......Page 26 Integral proteins......Page 27 Carbohydrates......Page 29 Glycolipids......Page 30 Glycoproteins......Page 32 Proteoglycans......Page 33 General features......Page 38 Membranes of prokaryotes......Page 39 Membranes of eukaryotes......Page 40 The surface (plasma) membrane......Page 42 Plasma membrane domains......Page 44 The cytoskeleton and glycocalyx......Page 46 Mitochondria and chloroplasts......Page 48 Peroxisomes (microbodies)......Page 51 Lysosomes......Page 52 Nuclei......Page 53 Endocytic compartment......Page 54 The secretory compartment......Page 55 References......Page 56 Homogenization......Page 58 Liquid shear......Page 59 Gaseous shear......Page 62 Other methods......Page 63 Homogenization of soft mammalian tissue......Page 64 Homogenization of tough tissues......Page 66 Homogenization of cultured mammalian cells......Page 67 Plant tissue......Page 69 Bacteria......Page 71 Fractionation of subcellular organelles......Page 73 Differential centrifugation......Page 74 Density gradient centrifugation......Page 75 Density perturbation......Page 77 Fractionation by immunoaffinity......Page 78 Fractionation by continuous flow electrophoresis......Page 81 Fractionation in aqueous two-phase systems......Page 83 Chemical......Page 84 Enzyme markers......Page 86 Expression of data......Page 90 Nuclei......Page 93 Resolving a light mitochondrial fraction......Page 94 Plasma membrane......Page 96 Membrane vesicles......Page 97 References......Page 99 Quantitative assay......Page 102 Separation of membrane proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)......Page 105 Western blotting (immunoblotting)......Page 110 Resolution of proteins by isoelectric focusing and two-dimensional (2D) electrophoresis......Page 112 Lipid extraction......Page 114 Analysis of phospholipids......Page 115 Separation and analysis of phospholipids by HPLC......Page 119 Analysis of phospholipid fatty acid......Page 120 Determination of cholesterol......Page 121 Separation and analysis of glycolipids......Page 123 Glycoprotein oligosaccharide analysis......Page 125 Preparation of a crude glycoprotein fraction......Page 126 Glycoprotein fractionation......Page 127 Chemical assays for carbohydrates......Page 129 Oligosaccharide sequence analysis......Page 130 References......Page 132 Chapter 5: Investigating the topology of membrane proteins......Page 134 Demonstration of the orientation of membrane vesicles......Page 135 Identification and separation of peripheral and integral proteins......Page 137 Identification of proteins anchored by phosphatidylinositol......Page 138 Identification of lipid-modified proteins......Page 141 General experimental criteria......Page 145 Probes for integral membrane proteins......Page 146 Hydropathy plots for prediction of membrane protein topography......Page 155 Structural analysis of membrane proteins......Page 156 References......Page 158 Determination of the organization of membrane phospholipids......Page 160 Multilamellar vesicles (MLVs)......Page 161 Black lipid membranes......Page 162 Determination of the transverse distribution of membrane phospholipids......Page 163 Chemical probes......Page 164 Phospholipases......Page 165 Phospholipid transfer proteins......Page 167 Assay based on prothrombinase......Page 169 Studies of lipid transport......Page 170 Investigation of the transmembrane movement of phospholipids......Page 171 Transport of phospholipids and cholesterol between intracellular membranes......Page 175 References......Page 176 Genetic approaches to studies of protein targetting......Page 178 Biochemical approaches to studies of protein targetting......Page 179 Steps in protein targetting......Page 180 Nuclear targetting......Page 181 Targetting to peroxisomes......Page 182 Targetting to mitochondria......Page 184 Targetting to the ER......Page 186 Retention in the ER lumen......Page 189 Transfer to the Golgi apparatus......Page 190 Targetting to lysosomes......Page 191 Transit of mannose-6-phosphatase receptors to and from the plasma membrane and Golgi membranes......Page 192 Establishing the topography and location of the membrane proteins of the ER, Golgi and plasma membrane......Page 193 Targetting in prokaryotes......Page 195 References......Page 196 Receptors and endocytosis......Page 198 Membrane receptors......Page 199 Electron microscopy......Page 200 Biochemical studies......Page 203 Reconstitution of vesicular transport......Page 205 Semi-intact cell systems......Page 208 Transfer vesicles......Page 209 Endoplasmic reticulum to Golgi (anterograde transport)......Page 211 References......Page 212 Index......Page 214 Membrane Analysis provides a comprehensive review of laboratory methods for membrane study, with an emphasis on isolating membranes, analysing their composition and architecture, and investigating membrane function. This book provides a comprehensive review of laboratory methods for membrane study, with an emphasis on isolating membranes, analysing their composition and architecture, and investigating membrane function.