Membrane analysis

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