Chaperonin Protocols

by Schneider, Christine

• ISBN13: 9780896037397
• ISBN10: 0896037398
• Format: Hardcover
• Copyright: 2000-02-01
• Publisher: Humana Pr Inc

Summary

Christine Schneider has assembled a unique collection of readily reproducible protocols for the study of chaperonins, intracellular proteins critical to many biological processes. Written by experienced investigators who have successfully honed their methods to a fineness, the protocols focus on the purification of chaperonins from different species along with their corresponding cofactors, and on chaperonin activity assays for in vivo as well as in vitro work. Many activity assays are given for GroEL, which can also be applied to mitochondrial Hsp60. There are also assays for the eukaryotic chaperonin TRiC and handy methods-for example, one for preparing labeled probes-that can be used for various purposes and prove helpful in numerous different procedures. Informative and highly practical, Chaperonin Protocols offers both novice and experienced investigators fast access to today's best and most productive chaperonin methods, all explained in step-by-step detail to ensure robust and reproducible results.

Table of Contents

Preface	p. v
Contributors	p. ix
Purification of Archaeal Chaperonin from Sulfolobus shibatae	p. 1
Purification of Hsp60 from Thermus thermophilus	p. 15
Purification of GroEL from an Overproducing E. coli Strain	p. 29
Purification of GroES from an Overproducing E. coli Strain	p. 41
Purification of the Gp31 Co-chaperonin from Bacteriophage T4	p. 51
Removing Trace Fluorescent Contaminants from GroEL Preparations	p. 63
Assembly and Disassembly of GroEL and GroES Complexes	p. 65
GroEL/GroES Interaction Assayed by Protease Protection	p. 71
Determination of Chaperonin Activity In Vivo	p. 75
Interaction of Nonnative Polypeptide Substrates with the Escherichia coli Chaperonin GroEL	p. 97
Prevention of Rhodanese Aggregation by the Chaperonin GroEL	p. 111
Refolding of Bovine Mitochondrial Rhodanese by Chaperonins GroEL and GroES	p. 117
Assay of Malate Dehydrogenase: A Substrate for the E. coli Chaperonins GroEL and GroES	p. 127
Assay of Chaperonin-Assisted Refolding of Citrate Synthase	p. 133
Purification of Yeast Mitochondrial Hsp60	p. 139
Preparation of Recombinant Human Hsp 10	p. 145
Purification of the Cytosolic Chaperonin TRiC from Bovine Testis	p. 153
Monitoring Actin Folding: Purification Protocols for Labeled Proteins and Binding to DNase I-Sepharose Beads	p. 161
Folding Assays: Assessing the Native Conformation of Proteins	p. 169
Purification of Prefoldin	p. 179
Purification of GimC from Saccharomyces cerevisiae	p. 185
Analysis of Eukaryotic Molecular Chaperone Complexes Involved in Actin Folding	p. 195
Index	p. 207
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