Essential cell biology: a pratical approach 2 Cell function
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| Format: | Book |
|---|---|
| Language: | German |
| Published: |
Oxford [u.a.]
Oxford Univ. Press
2003
|
| Edition: | 1. publ. |
| Series: | The practical approach series
[263] |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XVI, 235 S. Ill., graph. Darst. |
| ISBN: | 0199638330 0199638322 |
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MARC
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Record in the Search Index
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|---|---|
| adam_text | Contents
list of protocols page xi
Abbreviations xv
1 Enzyme activities 1
Keith F. Tipton
1 Introduction 3
2 Enzyme specificity and nomenclature 1
3 Determination of enzyme activity 1
Reaction progress curves 2
The effects of enzyme concentration and expression of activities 3
Conditions for activity measurements 4
4 The effects of substrate concentration 5
The Michaelis Menten relationship 5
Failure to obey the Michaelis Menten equation 5
5 Enzyme inhibitors 9
Reversible inhibitors 9
Tight binding inhibitors 26
Irreversible inhibitors 29
6 Behaviour in vivo 40
Recovery from irreversible inhibition 40
Effects of cellular processes 41
Inhibitor specificity 43
References 44
2 Gene expression 47
Luis Parada, Cem ElU, Miroslav Dundr, and Tom Misteli
1 Introduction 47
2 Indirect immunofluorescence 48
3 GFP in fixed or living cells 51
4 Nuclear matrix preparation 53
5 Visualization of transcription sites 55
6 Fluorescence in situ hybridization 59
7 Chromosome preparation and staining 67
8 Fluorescent chromosome painting 70
References 76
3 Regulation of the cell cycle 79
Diana M. Gitig and Andrew Koff
1 Introduction 79
2 Cell biology: detection of cell cycle arrests 79
3 Genetics: knockout and transgenic mice 83
4 Biochemistry 85
In vitro: recombinant proteins 85
In vivo: extract reconstitution assays 86
5 Conclusions 91
Acknowledgements 91
References 91
4 Cellular bioenergetics 95
David G. Nicholls and Monus W. Ward
1 Introduction 95
2 Mitochondrial membrane potential 95
Monitoring dynamic changes in mitochondrial membrane potential with TMRM+ or
rhodamine 123 97
Interpretation of dynamic whole cell fluorescence traces obtained with TMRM+ or
rhodamine 123 99
Equilibrium monitoring of mitochondrial membrane potential 99
Cytoplasmic free Ca2+ concentration with fura 2 101
Simultaneous monitoring with TMRM+ or rhodamine 123 of dynamic changes in
Ai|)m and cytoplasmic free calcium concentration 101
3 Monitoring mitochondrial superoxide generation 103
4 Monitoring hydrogen peroxide generation 104
5 ATP/ADP ratios in cultured neurons 104
References 105
5 Targeting of nuclear encoded proteins into and across the thylakoid
membrane: isolation and analysis of intact chloroplasts and thylakoids
from plants 107
Colin Robinson and Alexandra Mant
1 Introduction 107
Choice of plants 108
Growth conditions 108
2 Isolation of intact chloroplasts 108
3 In vitro synthesis of nuclear encoded chloroplast proteins 113
4 Import of proteins into isolated chloroplasts 114
5 Import of proteins into isolated thylakoid membranes 118
The import pathway for thylakoid lumenproteins 118
The basic import assay 119
Some variations on the basic assay 120
Analysis of nuclear encoded thylakoid membrane proteins 121
References 125
6 Transport into and out of the nucleus m
Patrizia Fanara, Adam C. Berger, Deanna M. Green, Henry Hagan, Michelle T. Harreman,
Kavita A. Marfatia, B. Booth Quimby, Maureen A Powers, and Anita H. Corbett
1 Introduction 127
2 Nuclear transport assays 128
In vitro protein import in permeabilized cells 128
In vivo NLS GFP protein import assay 132
Single nuclear pore transport assay 134
In vivo transport studies using microinjection into Xenopus oocytes 134
In vivo RNA localization using fluorescence in situ hybridization (FISH) 136
3 Nucleocytoplasmic protein shuttling assays 139
Heterokaryon assays 139
nup49 nuclear protein export assay in S. cerevisiae 141
Antibody injection to detect protein shuttling 142
4 Regulated nuclear transport: protein protein interactions 143
Ligand binding studies using overlay assays 143
Fluorescence based applications to map protein protein interactions 147
5 Genetic approaches to studying nuclear transport 148
References 149
7 Transport across the membrane of the endoplasmic reticulum 151
Karin Romisch
1 Introduction 151
2 Translocation into the ER and monitoring import 151
3 Interaction with chaperones and protein folding 158
4 Export of misfolded proteins from the ER to the cytosol for
degradation 160
References 162
8 In vitro reconstitutlon of early to late endosome transport: biogenesis
and subsequent fusion of transport Intermediates 165
Feng Gu and Jean Gruenberg
1 Introduction 165
2 Baby hamster kidney (BHK21) cell culture 167
3 Labelling of the different endosomal compartments in BHK cells 168
4 Homogenization and subcellular fractionation of BHK 170
5 In vitro reconstitution of the ECV/MVB formation from early
endosomes 172
6 In vitro fusion of the ECV/MVBs with late endosomes 176
7 Conclusion 179
References 179
9 Receptor biology 181
Mercedes Dosil, Pamela Mentesana, and James B. Konopka
1 Introduction 181
2 Ligand binding assays 183 ;
General considerations 181 :
Assaying ligand binding to membrane preparations 182 ¦
Assaying ligand binding with whole cells 184 ;
Data analysis 185
3 Epitope tags and fusion proteins 186
Protein detection tags 186
Chimeric receptors and GPCR Ga fusion proteins 187
4 Receptor modifications and receptor structure 188
Post translational modifications 188
Receptor structure 189
5 Receptor trafficking 190
Visualization of receptor trafficking and analysis of receptor loss from the plasma
membrane 191
Membrane fractionation 191
6 Heterologous expression of receptors in yeast 194
Background 194
General considerations 194
Growth and transformation of yeast 195
Analysis of receptor expression and activation 196
References 197
10 Measurement of signal transduction machinery 199
Matthew Hodgkin, Michael Wakelam, and Carolyn Armour
1 Introduction 199
2 Quantification of phospholipase C (PLC) signalling in cells 199
Measurement of PLC signal transduction pathway activity 200
Protein kinase C assay 205
Fluorescent quantification of changes in intracellular calcium concentration 207
3 Quantification of phospholipase D (PLD) activity in cells 208
Measurement of DAG and ceramide mass 209
4 Phosphatidylinositol 3 kinase (PI 3 kinase) signalling in cells 211
Measurement of PI 3 kinase downstream targets 212
Quantification of 3 phosphorylated lipids in cells 213
5 Measurement of small G protein function in cells 214
Additional notes on the measurement of SMG protein activation 216
6 Measurement of MAP kinase activation 217
ERK kinase activity 217
7 cAMP signalling in cells 219
8 Changes in cellular localization during signalling 220
Changes in cytoskeletal architecture 222
Visualization of signal transduction events in living cells 223
References 223
Al List of suppliers 225
Index 233
|
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| illustrated | Illustrated |
| indexdate | 2024-07-09T19:16:14Z |
| institution | BVB |
| isbn | 0199638330 0199638322 |
| language | German |
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| physical | XVI, 235 S. Ill., graph. Darst. |
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| publisher | Oxford Univ. Press |
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| series | The practical approach series |
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| spelling | Essential cell biology a pratical approach 2 Cell function ed. by John Davey ... 1. publ. Oxford [u.a.] Oxford Univ. Press 2003 XVI, 235 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series [263] The practical approach series ... Cell Physiology Cytology Laboratory manuals Molecular Biology Davey, John Sonstige oth (DE-604)BV017291516 2 The practical approach series [263] (DE-604)BV007921321 263 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010423393&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Essential cell biology a pratical approach The practical approach series Cell Physiology Cytology Laboratory manuals Molecular Biology |
| title | Essential cell biology a pratical approach |
| title_auth | Essential cell biology a pratical approach |
| title_exact_search | Essential cell biology a pratical approach |
| title_full | Essential cell biology a pratical approach 2 Cell function ed. by John Davey ... |
| title_fullStr | Essential cell biology a pratical approach 2 Cell function ed. by John Davey ... |
| title_full_unstemmed | Essential cell biology a pratical approach 2 Cell function ed. by John Davey ... |
| title_short | Essential cell biology |
| title_sort | essential cell biology a pratical approach cell function |
| title_sub | a pratical approach |
| topic | Cell Physiology Cytology Laboratory manuals Molecular Biology |
| topic_facet | Cell Physiology Cytology Laboratory manuals Molecular Biology |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010423393&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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