Analysis of RNA protein complexes in vitro:
Gespeichert in:
| Hauptverfasser: | , , |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
Amsterdam [u.a.]
Elsevier
1998
|
| Schriftenreihe: | Laboratory techniques in biochemistry and molecular biology
26 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | X, 237 S. Ill., graph. Darst. |
| ISBN: | 0444824197 0444824189 |
Internformat
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| 100 | 1 | |a Kjems, Jørgen |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Analysis of RNA protein complexes in vitro |c J/orgen Kjems, Jan Egebjerg and Jan Christiansen |
| 246 | 1 | 3 | |a Analysis of RNA-protein complexes in vitro |
| 264 | 1 | |a Amsterdam [u.a.] |b Elsevier |c 1998 | |
| 300 | |a X, 237 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 650 | 7 | |a RNA |2 gtt | |
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Datensatz im Suchindex
| _version_ | 1804126789366185984 |
|---|---|
| adam_text | Contents
Preface v
Chapter 1. Introduction to RNA protein interactions 1
1.1. Introduction 1
1.2. RNA secondary structures 1
1.2.1. Helices 1
1.2.2. Bulges and internal loops 2
1.2.3. Hairpin loops 4
1.2.4. Single stranded regions 5
1.3. RNA tertiary structures 5
1.3.1. Triple base pairs (single strand/helix interactions) 5
1.3.2. Pseudoknots (single strand/single strand interaction) 5
1.3.3. Hairpin/internal loop interactions 6
1.4. Protein motifs involved in RNA binding 6
1.4.1. Ribonucleoprotein (RNP) motif 6
1.4.2. The K homology domain (KH) 8
1.4.3. The arginine rich motif (ARM) 9
1.4.4. The RGG box 9
1.4.5. Zinc fingers 10
1.4.6. Double strand RNA binding domains (dsRBD) 10
References 11
Chapter 2. Preparation of RNA 13
2.1. Working with RNA 13
2.1.1. Special precautions when working with RNA 13
2.1.2. Basic protocols for RNA work 14
2.1.2.1. RNA quantification 14
2.1.2.2. Gel purification 15
2.1.2.3. Extraction with organic solvents 16
2.1.2.4. Precipitation 16
2.1.2.5. Storage of RNA 17
2.1.2.6. RNA renaturation 17
vii
viii
2.1.2.7. Desalting and removal of nucleotides 18
2.1.2.8. Diethylpyrocarbonate 18
2.1.2.9. Standard reagents 18
2.2. Isolation of RNA from cells 19
2.2.1. Guanidinium thiocyanate—CsCl method 20
2.2.2. Single step guanidinium thiocyanate acid phenol method .... 23
2.2.3. Hot phenol SDS method 25
2.2.4. Vanadyl ribonucleoside complex method for preparation of
nuclear and cytoplasmic RNA 27
2.2.5. Purification of poly A+ RNA 29
2.3. In vitro synthesis of RNA 32
2.3.1. In vitro transcription of RNA 33
2.3.1.1. Quantification of in vitro transcribed RNA (in the
presence of trace amount of a [32P] UTP) 35
2.3.2. Co transcriptional labelling or modification of RNA 35
2.3.2.1. Generation of specific terminus by RNase H 41
2.3.2.2. Cotranscriptional incorporation of nucleotides
containing modified ribose moieties 40
2.3.3. End labelling of RNA 43
2.3.3.1. Dephosphorylation of RNA 43
2.3.3.2. 5 end labeling of RNA 45
2.3.3.3. 3 end labeling of RNA 46
2.3.3.3.1. RNA ligase method 46
2.3.3.3.2. Poly (A) polymerase method 46
2.3.3.4. Preparation of [a32P] pCp from Cp and [y i2P]
ATP 47
2.3.4. Specific modification of RNA at an internal site 48
References 53
Chapter 3. Preparation of protein 57
3.1. Isolation of protein from cells 57
3.1.1. Preparation of HeLa cell extract 57
3.1.2. Purification of SR proteins from nuclear extract 62
3.2. Expression and purification of recombinant proteins in E. coli 65
3.2.1. Expression of recombinant proteins in E. coli 66
3.2.2. Purification of GST tagged proteins under native conditions. . 69
3.2.3. Purification of His tagged proteins under denaturing
conditions 72
3.2.4. Labeling of protein using heart muscle kinase 74
3.3. In vitro translation 76
References 79
ix
Chapter 4. Preparation and analysis of RNA protein
complexes in vitro 83
4.1. Formation of RNA protein complexes 83
4.2. Analysis of RNA protein complexes 84
4.2.1. Filter binding assay 85
4.2.2. Mobility shift analysis 88
4.2.3. Sucrose gradient analysis 92
4.3. Isolation and identification 96
4.3.1. Affinity purification using tagged RNA or protein 96
4.3.1.1. Binding of biotinylated protein or RNA to
streptavidin beads 99
4.3.1.2. Binding of protein to glutathione beads 101
4.3.1.3. Affinity purification of RNA protein complexes .... 102
4.3.2. UV cross linking 105
4.4. RNA footprinting 110
4.4.1. Enzymatic probing 114
4.4.2. Chemical probing 117
4.4.2.1. DMS (dimethylsulphate) modification for probing
adenosines and cytidines 119
4.4.2.2. DMS (dimethylsulphate) modification for probing
guanosines 122
4.4.2.3. DEP (diethylpyrocarbonate) modification for
probing adenosines 123
4.4.2.4. Kethoxal (3 ethoxy 2 oxo butanal) modification for
probing guanosines 125
4.4.2.5. CMCT (l cycIohexyl 3 (2 morpholinoethyl)
carbodiimide for probing uridines and guanosines . . 126
4.4.2.6. Hydroxyl radical modification for probing ribose
moieties 127
4.4.2.7. Iodine scission of phosphorothioate containing
RNA for probing phosphates 129
4.4.3. Identification of modified residues 130
4.4.3.1. Reverse transcriptase assay 130
4.4.3.1.1. Extension from an end labelled primer. 133
4.4.3.1.2. Extension in the presence of radioactive
dATP 134
4.4.3.1.3. Sequencing by the reverse transcriptase
method 136
4.4.3.2. End labelling method 137
4.4.3.2.1. Enzymatic sequencing of end labelled
RNA 139
4.4.3.2.2. Chemical sequencing of end labelled
RNA 142
X
4.5. Protein footprinting 147
4.5.1. Protein footprinting using proteinases 150
4.5.2. Protein footprinting using Fe2+/H2O2 157
4.5.3. Preparation of Tris/Tricine SDS polyacrylamide gels 159
4.6. Modification interference 161
4.6.1. Base modifications 163
4.6.2. Phosphate modification by ethylnitrosourea (ENU) 164
4.6.3. Analogue interference 168
4.7. SELEX 169
References 176
Chapter 5. Functional analysis of RNA protein complexes
in vitro 181
5.1. Pre mRNA splicing 181
5.1.1. In vitro mRNA splicing 182
5.1.2. Analysis of RNA splicing products by denaturing gel
electrophoresis 185
5.1.3. Analysis of RNA splicing complexes by native gel
electrophoresis 189
5.1.4. Debranching of RNA lariats 191
5.1.5. Analysis of RNA splicing complexes by sucrose gradients. . . . 193
5.1.6. Affinity purification and characterization of splicosome
complexes 197
5.2. Polyadenylation 204
5.2.1. In vitro polyadenylation 204
5.2.2. Analysis of mRNA polyadenylation states 207
5.3. RNA modification 210
5.3.1. Reverse transcriptase polymerase chain reaction (RT PCR) . . 213
5.3.2. Primer extension by Klenow polymerase (after RT PCR) .... 218
5.3.3. Primer extension by reverse transcriptase 219
5.4. Translation 220
5.4.1. Preparation of ribosomes 220
5.4.1.1. Bacterial ribosomes 221
5.4.1.2. Mammalian ribosomes 223
5.4.2. Preparation and analysis of polysomes 225
References 231
|
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| series2 | Laboratory techniques in biochemistry and molecular biology |
| spelling | Kjems, Jørgen Verfasser aut Analysis of RNA protein complexes in vitro J/orgen Kjems, Jan Egebjerg and Jan Christiansen Analysis of RNA-protein complexes in vitro Amsterdam [u.a.] Elsevier 1998 X, 237 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Laboratory techniques in biochemistry and molecular biology 26 Eiwitten gtt Interactie gtt RNA gtt Proteins analysis Laboratory Manuals RNA analysis Laboratory Manuals RNA-protein interactions Methode (DE-588)4038971-6 gnd rswk-swf In vitro (DE-588)4250701-7 gnd rswk-swf RNS-Bindungsproteine (DE-588)4178253-7 gnd rswk-swf RNS-Bindungsproteine (DE-588)4178253-7 s Methode (DE-588)4038971-6 s DE-604 In vitro (DE-588)4250701-7 s Egebjerg, Jan Verfasser aut Christiansen, Jan Verfasser aut Laboratory techniques in biochemistry and molecular biology 26 (DE-604)BV005871299 26 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008249194&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kjems, Jørgen Egebjerg, Jan Christiansen, Jan Analysis of RNA protein complexes in vitro Laboratory techniques in biochemistry and molecular biology Eiwitten gtt Interactie gtt RNA gtt Proteins analysis Laboratory Manuals RNA analysis Laboratory Manuals RNA-protein interactions Methode (DE-588)4038971-6 gnd In vitro (DE-588)4250701-7 gnd RNS-Bindungsproteine (DE-588)4178253-7 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4250701-7 (DE-588)4178253-7 |
| title | Analysis of RNA protein complexes in vitro |
| title_alt | Analysis of RNA-protein complexes in vitro |
| title_auth | Analysis of RNA protein complexes in vitro |
| title_exact_search | Analysis of RNA protein complexes in vitro |
| title_full | Analysis of RNA protein complexes in vitro J/orgen Kjems, Jan Egebjerg and Jan Christiansen |
| title_fullStr | Analysis of RNA protein complexes in vitro J/orgen Kjems, Jan Egebjerg and Jan Christiansen |
| title_full_unstemmed | Analysis of RNA protein complexes in vitro J/orgen Kjems, Jan Egebjerg and Jan Christiansen |
| title_short | Analysis of RNA protein complexes in vitro |
| title_sort | analysis of rna protein complexes in vitro |
| topic | Eiwitten gtt Interactie gtt RNA gtt Proteins analysis Laboratory Manuals RNA analysis Laboratory Manuals RNA-protein interactions Methode (DE-588)4038971-6 gnd In vitro (DE-588)4250701-7 gnd RNS-Bindungsproteine (DE-588)4178253-7 gnd |
| topic_facet | Eiwitten Interactie RNA Proteins analysis Laboratory Manuals RNA analysis Laboratory Manuals RNA-protein interactions Methode In vitro RNS-Bindungsproteine |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008249194&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV005871299 |
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