Ubiquitin-Proteasome Protocols

by Patterson, Cam; Cyr, Douglas M.

• ISBN13: 9781588292520
• ISBN10: 1588292525
• Format: Hardcover
• Copyright: 2005-04-15
• Publisher: Humana Pr Inc

Summary

A collection of cutting-edge techniques for studying ubiquitin-dependent protein degradation via the proteasome. The topics covered range broadly from basic biochemistry to cellular assays to discovery techniques using mass spectrometric analysis. These biochemical and cellular methods are necessary to explore the ubiquitin-proteasome system and ubiquitin-proteasome-dependent functions. State-of-the-art and user-friendly, Ubiquitin-Proteasome Protocols offers novice and experienced bench scientists alike a thorough compendium of readily reproducible techniques that will accelerate discovery, enhance productivity, and permit manipulation of the system for varied research purposes.

Table of Contents

Preface	p. v
Contributors	p. ix
Biochemical Methods to Study the Ubiquitin-Proteasome System
Small-Molecule Inhibitors of Proteasome Activity	p. 3
Purification of E1 and E1-Like Enzymes	p. 23
Assays for RING Family Ubiquitin Ligases	p. 37
Ubiquitin Chain Synthesis	p. 47
Purification of Proteasomes, Proteasome Subcomplexes, and Proteasome-Associated Proteins From Budding Yeast	p. 57
Recognition and Processing of Misfolded Proteins by PA700, the 19S Regulatory Complex of the 26S Proteasome	p. 71
Cell-Free Assay for Ubiquitin-Independent Proteasomal Protein Degradation	p. 83
Assays of Proteasome-Dependent Cleavage Products	p. 97
Identification of Components of Protein Complexes	p. 117
Mass Spectrometric Determination of Protein Ubiquitination	p. 153
Reconstitution of Endoplasmic Reticulum-Associated Degradation Using Yeast Membranes and Cytosol	p. 175
Reticulocyte Lysate as a Model System to Study Endoplasmic Reticulum Membrane Protein Degradation	p. 185
Deubiquitinating Enzyme Purification, Assay Inhibitors, and Characterization	p. 207
Cellular Methods to Study Ubiquitin-Proteasome-Dependent Functions
Measuring Ubiquitin Conjugation in Cells	p. 223
Assays for Proteasome Assembly and Maturation	p. 243
N-Terminal Ubiquitination	p. 255
Quantitating Defective Ribosome Products	p. 271
Endoplasmic Reticulum-Associated Protein Quality Control and Degradation: Screen for ERAD Mutants After Ethylmethane Sulfonate Mutagenesis	p. 283
Endoplasmic Reticulum-Associated Protein Quality Control and Degradation: Genome Wide Screen for ERAD Components	p. 289
Cystic Fibrosis Transmembrane Conductance Regulator as a Model Substrate to Study Endoplasmic Reticulum Protein Quality Control in Mammalian Cells	p. 293
Aggresome Formation	p. 305
Detection of Sumoylated Proteins	p. 329
Proteasome Inhibitors in Cancer Therapy	p. 339
Parkinson's Disease: Assays for the Ubiquitin Ligase Activity of Neural Parkin	p. 351
Index	p. 371
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