Translation initiation : cell biology, high-throughput methods, and chemical-based approaches / edited by Jon Lorsch.

Format:

Book

Contributor:

Lorsch, Jon.

Series:

Methods in enzymology ; v. 431.

Methods in enzymology, 0076-6879 ; v. 431

Language:

English

Subjects (All):

Genetic translation.

Genetic regulation.

Physical Description:

1 online resource (393 p.)

Place of Publication:

San Diego, Calif : Academic Press, c2007.

Language Note:

English

Summary:

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.

Contents:

Front Cover; Translation Initiation: Cell Biology, High-Throughput Methods, and Chemical-Based Approaches; Copyright Page; Contetns; Contributors; Preface; Volumes in Series; Chapter 1: Purification of FLAG-Tagged Eukaryotic Initiation Factor 2B Complexes, Subcomplexes, and Fragments from Saccharomyces Cerevisiae; 1. Introduction; 2. Plasmid Vectors and Yeast Strains Used; 3. Expression and Purification of eIF2B; 3.1. Buffers for protein purification; 3.2. Cell growth and harvest; 3.3. Cell lysis; 3.4. Purification using anti-FLAG M2 affinity resin; 3.5. Dialysis to remove 3XFLAG peptide

4. Functional Analysis of Purified Proteins5. Conclusions; Acknowledgments; References; Chapter 2: In Vivo Deletion Analysis of the Architecture of a Multiprotein Complex of Translation Initiation Factors; 1. Introduction; 2. Ni2+ Affinity Purification of eIF3 Using a Polyhistidine Tag; 2.1. Affinity-tagging; 2.2. Whole-cell extract (WCE) preparation; 2.3. Batch Ni2+ affinity purification of eIF3 (Ni pull-down [Ni PD]); 2.4. Analysis of the purified proteins; 2.5. Troubleshooting; 3. Deletion/Mutational Analysis of eIF3 Subunits and Ni2+ Affinity Purification of Their Subcomplexes

3.1. Typical example4. eIF3 Purification Using Other Epitope Tags; 4.1. Immunoaffinity purification of eIF3 containing hemagglutinin-tagged eIF3i/TIF34 (HA pull-down); 4.2. Immunoaffinity purification of eIF3 containing FLAG-tagged eIF3g/TIF35 (FLAG pull-down); 5. CAM (Clustered-10-Alanine Mutagenesis); Acknowledgments; References; Chapter 3: An Approach to Studying the Localization and Dynamics of Eukaryotic Translation Factors in Live Yeast Cells; 1. Introduction; 1.1. Epitope tagging; 1.2. Amplification of epitope tagging cassette; 2. Yeast Strains and Growth Conditions

2.1. Yeast transformation2.2. Confirmation of epitope-tagged proteins; 2.3. PCR analysis to verify tagged genes; 2.4. Live cell imaging; 3. Application of Live Cell Imaging; 3.1. FRAP analysis; 3.2. Microscopy and FRAP analysis; 3.3. Analysis of FRAP experiments; 3.4. RNA localization; 4. Additional In Vivo Techniques; 5. Conclusions; Acknowledgments; References; Chapter 4: In Vitro and Tissue Culture Methods for Analysis of Translation Initiation on the Endoplasmic Reticulum; 1. Background; 2. Methods; 2.1. In vitro analysis of protein synthesis initiation on membrane-bound ribosomes

2.2. In vitro translation2.3. Ribosomal assembly status-ER membrane-restricted initiation; 3. Analysis of Protein Synthesis Initiation on ER Membrane-Bound Ribosomes of Tissue Culture Cells; 3.1. Background; 3.2. Modulating polyribosome loading; 3.3. Sequential detergent extraction; 4. Velocity Sedimentation; 4.1. Reagents; 4.2. Velocity sedimentation; Acknowledgments; References; Chapter 5: Mammalian Stress Granules and Processing Bodies; 1. Introduction; 2. Detection and Identification of SGs and PBs; 3. Immunostaining Protocol; 3.1. Buffers and solutions; 3.2. Staining procedure

4. Stimuli for the Induction of SGs and PBs

Notes:

Description based upon print version of record.

Includes bibliographical references and indexes.

ISBN:

9786611059446

9781281059444

1281059447

9780080553481

0080553486

OCLC:

476113024