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Methods in Enzymology, Volume 431 : Cell Biology, High-throughput and Chemical-based Approaches: Volume 431 - Jon Lorsch

Methods in Enzymology, Volume 431

Cell Biology, High-throughput and Chemical-based Approaches: Volume 431

Hardcover Published: 12th October 2007
ISBN: 9780123739643
Number Of Pages: 392

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For over fifty years the Methods in Enzymology series has been the critically aclaimed laboratory standard and one of the most respected publications in the field of biochemistry. The highly relevant material makes it an essential publication for researchers in all fields of life and related sciences. This volume, the third of three on the topic of Translation Initiation includes articles written by leaders in the field.

Contributorsp. xi
Prefacep. xvii
Volumes in Seriesp. xix
Purification of FLAG-Tagged Eukaryotic Initiation Factor 2B Complexes, Subcomplexes, and Fragments from Saccharomyces cerevisiaep. 1
Introductionp. 2
Plasmid Vectors and Yeast Strains Usedp. 4
Expression and Purification of elF2Bp. 6
Functional Analysis of Purified Proteinsp. 10
Conclusionsp. 10
Acknowledgmentsp. 10
Referencesp. 11
In Vivo Deletion Analysis of the Architecture of a Multiprotein Complex of Translation Initiation Factorsp. 15
Introductionp. 16
Ni[superscript 2+] Affinity Purification of elF3 Using a Polyhistidine Tagp. 21
Deletion/Mutational Analysis of elF3 Subunits and Ni[superscript 2+] Affinity Purification of Their Subcomplexesp. 24
elF3 Purification Using Other Epitope Tagsp. 27
CAM (Clustered-30-Alanine Mutagenesis)p. 29
Acknowledgmentsp. 30
Referencesp. 31
An Approach to Studying the Localization and Dynamics of Eukaryotic Translation Factors in Live Yeast Cellsp. 33
Introductionp. 34
Yeast Strains and Growth Conditionsp. 37
Application of Live Cell Imagingp. 39
Additional In Vivo Techniquesp. 43
Conclusionsp. 44
Acknowledgmentsp. 44
Referencesp. 44
In Vitro and Tissue Culture Methods for Analysis of Translation Initiation on the Endoplasmic Reticulump. 47
Backgroundp. 48
Methodsp. 49
Analysis of Protein Synthesis Initiation on ER Membrane-Bound Ribosomes of Tissue Culture Cellsp. 53
Velocity Sedimentationp. 57
Acknowledgmentsp. 59
Referencesp. 59
Mammalian Stress Granules and Processing Bodiesp. 61
Introductionp. 62
Detection and Identification of SGs and PBsp. 63
Immunostaining Protocolp. 70
Stimuli for the Induction of SGs and PBsp. 76
Transfection-lnduced SGsp. 77
Induction of PBsp. 78
Conclusionsp. 79
Referencesp. 79
Methods to Analyze MicroRNA-mediated Control of mRNA Translationp. 83
Introductionp. 84
Systems to Measure miR-Mediated Repressionp. 85
Detection of miR-Mediated Changes in mRNA Stabilityp. 93
Measurement of miR Target mRNA Deadenylationp. 97
Measuring the Distribution of miRNP Components in Polysome Profilesp. 99
Localization of miRNP Components to P-Bodiesp. 105
Concluding Remarksp. 108
Acknowledgmentsp. 108
Referencesp. 108
Methods for Studying Signal-Dependent Regulation of Translation Factor Activityp. 113
Introduction to Signaling Pathways and the Control of Translation Factorsp. 114
Experimental Protocolsp. 126
Analysis of the Phosphorylation States of Translation Factors and Signaling Components - Overviewp. 128
Assays for Specific Protein Kinasesp. 132
Assays for Initiation Factor Functionp. 135
Referencesp. 138
Analysis of mRNA Translation in Cultured Hippocampal Neuronsp. 143
Materials and Reagents for Primary Neuron Culturep. 144
RNA Transfection and Reporter Assaysp. 149
shRNA Design and Lenti-shRNA Virus Productionp. 153
shRNA Knockdown of CPEB3 in Cultured Neuronsp. 157
Synaptoneurosome Isolation and [superscript 35]S-met/cys Labelingp. 158
UV-Crosslinking, Immunoprecipitation of an RNA-Binding Protein, CPEB3p. 159
Referencesp. 161
Detecting Ribosomal Association with the 5[prime] Leader of mRNAs by Ribosome Density Mapping (RDM)p. 163
Introductionp. 164
General Conceptp. 164
Methodsp. 168
Acknowledgmentsp. 174
Referencesp. 174
Genome-Wide Analysis of mRNA Polysomal Profiles with Spotted DNA Microarraysp. 177
Introductionp. 178
Experimental Designsp. 179
Methodsp. 189
Data Acquisition and Analysisp. 199
Acknowledgmentsp. 200
Referencesp. 201
Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Transtation and Stabilityp. 203
Introductionp. 204
Chemical Synthesisp. 207
In Vitro and In Vivo Assaysp. 213
Binding Affinity of ARCAs for elF4Ep. 214
Incorporation of ARCAs into RNA by In Vitro Transcriptionp. 214
Properties of ARCAs and ARCA-Capped mRNAs in Cell-Free Translation Systemsp. 219
Properties of ARCA-Capped mRNAs in Mammalian Cellsp. 221
Acknowledgmentsp. 225
Referencesp. 225
Methods for Identifying Compounds that Specifically Target Translationp. 229
Introductionp. 230
Materials Requiredp. 231
Methods and Testsp. 240
Acknowledgmentsp. 264
Referencesp. 264
Identifying Small Molecule Inhibitors of Eukaryotic Translation Initiationp. 269
Introductionp. 270
A Reverse Chemical Genetic Assay Probing elF4E:elF4G and elF4A:efF4G Interactionsp. 275
A Forward Chemical Genetic Screen to Identify Inhibitors of Eukaryotic Translationp. 285
Characterization of Inhibitors of Translation Identified in Chemical Genetic Screensp. 291
Discussion and Concluding Remarksp. 296
Acknowledgmentsp. 297
Referencesp. 297
Isolation and Identification of Eukaryotic Initiation Factor 4A as a Molecular Target for the Marine Natural Product Pateamine Ap. 303
Introductionp. 304
Total Synthesis of Pateamine Ap. 307
Synthesis of Derivatives of Pateamine A and Structure Activity Relationship Studiesp. 309
Structural Analysis of PatA Leading to DMDA-PatA and Viable Positions for Derivatizationp. 313
Synthesis of a Bioactive Biotin-Pateamine A (B-PatA) Conjugatep. 313
Affinity Pull-Down of PatA-Binding Proteinsp. 315
Identification of PatA-Binding Proteinsp. 319
Concluding Remarksp. 321
Acknowledgmentsp. 321
Referencesp. 322
Author Indexp. 325
Subject Indexp. 343
Table of Contents provided by Ingram. All Rights Reserved.

ISBN: 9780123739643
ISBN-10: 0123739640
Series: Methods in Enzymology
Audience: Professional
Format: Hardcover
Language: English
Number Of Pages: 392
Published: 12th October 2007
Publisher: Elsevier Science Publishing Co Inc
Country of Publication: US
Dimensions (cm): 22.9 x 15.2  x 2.82
Weight (kg): 0.86
Edition Number: 431

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