Buch, Englisch, 432 Seiten, Format (B × H): 195 mm x 255 mm, Gewicht: 1072 g
ISBN: 978-0-470-84758-9
Verlag: Wiley
As a modern composite scientific discipline, Cell Biology has expanded and moved forward rapidly in recent years. Cell Biologists now require a wide range of techniques, including those of analytical biochemistry and microscopy in all its diverse forms. These are often used alongside the techniques of molecular biology and molecular genetics. This book contains numerous useful protocols, covering light and electron microscopy, cell culture, cell separation, subcellular fractionation, organelle and membrane isolation, and the use of in vitro reassembly systems in Cell Biology. Many of these protocols feature helpful notes and safety information for practical application. The format favours easy use at the bench with space for notes and important safety information. An appendix contains essential analytical information that will prove invaluable to those working on all aspects of cell biology.
This book will be of interest to students and more experienced cell biologists, as well as molecular biologists and those working in genomics and proteomics who are looking for cellular techniques to validate their findings within intact cells.
Autoren/Hrsg.
Fachgebiete
Weitere Infos & Material
Preface xi
List of Contributors xiii
1 Basic Light Microscopy 1
Minnie O’Farrell
Introduction 1
Key components of the compound microscope 2
Techniques of microscopy 6
Protocols
1.1 Setting up the microscope for bright field microscopy 7
1.2 Setting K¨ohler illumination 8
1.3 Focusing procedure 9
1.4 Setting up the microscope for phase contrast microscopy 11
1.5 Setting up the microscope for epifluorescence 14
1.6 Poly-L-lysine coating 18
References 19
2 Basic Electron Microscopy 21
J. Robin Harris
Introduction 21
EM methods available 22
Protocols
2.1 Preparation of carbon-formvar, continuous carbon and holey carbon support films 25
2.2 The ‘droplet’ negative staining procedure (using continuous carbon, formvar–carbon and holey carbon support films) 27
2.3 Immunonegative staining 29
2.4 The negative staining-carbon film technique: cell and organelle cleavage 31
2.5 Preparation of unstained and negatively stained vitrified specimens 33
2.6 Metal shadowing of biological specimens 35
2.7 A routine schedule for tissue processing and resin embedding 37
2.8 Agarose encapsulation for cell and organelle suspensions 39
2.9 Routine staining of thin sections for electron microscopy 40
2.10 Post-embedding indirect immunolabelling of thin sections 42
2.11 Imaging the nuclear matrix and cytoskeleton by embedment-free electron microscopy 44
Jeffrey A. Nickerson and Jean Underwood
References 50
3 Cell Culture 51
Anne Wilson and John Graham
Cells: isolation and analysis 51
Anne Wilson
Mechanical disaggregation of tissue 52
Protocols
3.1 Tissue disaggregation by mechanical mincing or chopping 54
3.2 Tissue disaggregation by warm trypsinization 56
3.3 Cold trypsinization 58
3.4 Disaggregation using collagenase or dispase 60
Anne Wilson
3.5 Recovery of cells from effusions 63
Anne Wilson
3.6 Removal of red blood cells by snap lysis 64
3.7 Removal of red blood cells and dead cells using isopycnic centrifugation 65
Anne Wilson
3.8 Quantitation of cell counts and viability 67
Anne Wilson
3.9 Recovery of cells from monolayer cultures 71
Anne Wilson
3.10 Freezing cells 74
3.11 Thawing cells 76
John Graham
3.12 Purification of human PBMCs on a density barrier 80
3.13 Purification of human PBMCs using a mixer technique 82
3.14 Purification of human PBMCs using a barrier flotation technique 83
References 84
4 Isolation and Functional Analysis of Organelles 87
John Graham
Introduction 88
Homogenization 88
Differential centrifugation 90
Density gradient centrifugation 91
Nuclei and nuclear components 92
Mitochondria 93
Lysosomes 94
Peroxisomes 94
Rough and smooth endoplasmic reticulum (ER) 95
Golgi membranes 96
Plasma membrane 96
Chloroplasts 97
Protocols
4.1 Isolation of nuclei from mammalian liver in an iodixanol gradient (with notes on cultured cells) 98
4.2 Isolation of metaphase chromosomes 100
4.3 Isolation of the nuclear envelope 102
4.4 Nuclear pore complex isolation 104
J. Robin Harris
4.5 Preparation of nuclear matrix 106
4.6 Preparation of nucleoli 107
4.7 Isolation of a heavy mitochondrial fraction from rat liver by differential centrifugation 108
4.8 Preparation of a light mitochondrial fraction from tissues and cultured cells 110
4.9 Purification of yeast mitochondria in a discontinuous Nycodenz® gradient 112
4.10 Purification of mitochondria from mammalian liver or cultured cells in a median-loaded discontinuous Nycodenz® gradient 114
4.11 Succinate–INT reductase assay 116
4.12 Isolation of lysosomes in a discontinuous Nycodenz® gradient 117
4.13 ß-Galactosidase (spectrophotometric assay) 119
4.14 ß-Galactosidase (fluorometric assay) 120
4.15 Isolation of mammalian peroxisomes in an iodixanol gradient 121
4.16 Catalase assay 123
4.17 Analysis of major organelles in a preformed iodixanol gradient 124
4.18 Separation of smooth and rough ER in preformed sucrose gradients 127
4.19 Separation of smooth and rough ER in a self-generated iodixanol gradient 129
4.20 NADPH-cytochrome c reductase assay 131
4.21 Glucose-6-phosphatase assay 132
4.22 RNA analysis 133
4.23 Isolation of Golgi membranes from liver 134
4.24 Assay of UDP-galactose galactosyl transferase 136
4.25 Purification of human erythrocyte ‘ghosts’ 137
4.26 Isolation of plasma membrane sheets from rat liver 139
4.27 Assay for 5’-nucleotidase 141
4.28 Assay for alkaline phosphodiesterase 143
4.29 Assay for ouabain-sensitive Na+/K+-ATPase 144
4.30 Isolation of chloroplasts from green leaves or pea seedlings 145
4.31 Measurement of chloroplast chlorophyll 147
4.32 Assessment of chloroplast integrity 148
5 Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling 153
John Graham
Introduction 154
Methods available 154
Plasma membrane domains 155
Analysis of membrane compartments in the endoplasmic reticulum–Golgi–plasma membrane pathway 156
Separation of membrane vesicles from cytosolic proteins 157
Endocytosis 158
Protocols
5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients 160
5.2 Isolation of rat liver sinusoidal domain using antibody-bound beads 162
5.3 Fractionation of apical and basolateral domains from Caco-2 cells in a sucrose gradient 163
5.4 Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient 165
5.5 Isolation of lipid rafts 167
5.6 Isolation of caveolae 170
5.7 Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose–D2O density gradients 172
5.8 Analysis of Golgi, ER, ERGIC and other membrane compartments from cultured cells using continuous iodixanol density gradients 174
5.9 Analysis of Golgi, ER, TGN and other membrane compartments in sedimentation velocity iodixanol density gradients (continuous or discontinuous) 177
5.10 SDS–PAGE of membrane proteins 180
5.11 Semi-dry blotting 182
5.12 Detection of blotted proteins by enhanced chemiluminescence (ECL) 183
5.13 Separation of membranes and cytosolic fractions from (a) mammalian cells and (b) bacteria 185
5.14 Analysis of early and recycling endosomes in preformed iodixanol gradients; endocytosis of transferrin in transfected MDCK cells 188
5.15 Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver 191
5.16 Polysucrose–Nycodenz® gradients for the analysis of dense endosome–lysosome events in mammalian liver 194
References 196
6 In Vitro Techniques 201
Edited by J. Robin Harris
Introduction 203
Protocols
Nuclear components
6.1 Nucleosome assembly coupled to DNA repair synthesis using a human cell free system 204
Geneviève Almouzni and Doris Kirschner
6.2 Single labelling of nascent DNA with halogenated thymidine analogues 210
Daniela Dimitrova
6.3 Double labelling of DNA with different halogenated thymidine analogues 214
6.4 Simultaneous immunostaining of proteins and halogen-dU-substituted DNA 217
6.5 Uncovering the nuclear matrix in cultured cells 220
Jeffrey A. Nickerson, Jean Underwood and Stefan Wagner
6.6 Nuclear matrix–lamin
