Ubiquitin and Protein Degradation, Part A

Edited by

  • Raymond Deshaies, Howard Hughes Medical Institute, Caltech, Pasadena, CA, USA

Since the inception of the series, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. The series contains much material still relevant today - truly an essential publication for researchers in all field of life sciences. Ubiquitin and Protein Degradation, Part A will cover high level purification, bioinformatics analysis and substrate identification of the major proteins involved in protein degradation. The chapters are highly methodological and focus primarily on purification and analysis.
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Biochemists, biophysicists, cell biologists, molecular biologists, geneticists, developmental biologists


Book information

  • Published: August 2005
  • ISBN: 978-0-12-182803-5


se for the Series: "The Methods in Enzymology series represents the gold-standard."

"It is a true 'methods' series, including almost every detail from basic theory to sources of equipment and reagents, with timely documentation provided on each page."

Table of Contents

Section I – E1 EnzymesCh 1 High level expression and purification of recombinant E1 enzymeCh 2 Expression, purification and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complexCh 3 A FRET-based assay to study SUMO modification in solutionCh 4 Properties of the ISG15 E1 enzyme UbE1LSection II – E2 EnzymeCh 5 Purification and properties of the Ubiquitin Conjugating Enzymes Cdc34 and Ubc13-Mms2Ch 6 Eexpression, purification, and properties of Ubc4/5 family E2 enzymesCh 7 In vitro systems for Nedd8 conjugation by Ubc12Ch 8 Purification and activity assays for Ubc9, the ubiquitin conjugating enzyme for the small ubiquitin-like modifier SUMOCh 9 High-througput assay to monitor Formation of the E2-Ubiquitin thioester intermediateSection III – E3 EnzymeCh 10 Expression and Evaluation of RING Finger proteinsCh 11 Expression and assay of HECT domain ligasesCh 12 High Level Expression and Purification of Recombinant SCF Ubiquitin LigaseCh 13 In vitro reconstitution of SCF substrate ubiquitination with purified proteinsCh 14 Expression and assay of glycoprotein-specific ubiquitin ligasesCh 15 Affinity purification and assay of APC/C on p13 Suc1Ch 16 Large-Scale Purification of the Vertebrate Anaphase-Promoting Complex/CyclosomeCh 17 Purification and assay of the budding yeast Anaphase Promoting ComplexCh 18 Enzymology of the Anaphase-Promoting ComplexCh 19 Identification of Cell Cycle Dependent Phosphorylation Sites on the Anaphase-Promoting Complex/Cyclosome by Mass SpectrometryCh 20 Purification and assay of Mad2: A Two-state Inhibitor of APC/CCh 21 Identification, expression and assay of an oxidation-specific ubiquitin ligase, HOIL-1Ch 22 (In revision)Purification and assay of chaperon-dependent ubiquitin ligase CHIPCh 23 Methods for the functional genomic analysis of ubiquitin ligasesSection IV - ProteasomeCh 24 Purification of PA700, the 19S regulatory complex of the 26S ProteasomeCh 25 Purification and analysis of recombinant 11S acti vators of the 20S proteasome: Trypanosoma brucei PA26 and human PA28a, PA28B, and PA28yCh 26 Purification and assay of proteasome activator PA200Ch 27 Purification, crystallization and X-ray analysis of the yeast 20S proteasomeCh 28 (In revision due to figures only)Preparation of Hybrid (19S-20S-PA28) Proteasome Complexes and Analysis of Peptides Generated During Protein DegradationCh 29 Characterization of the Proteasome using Native Gel Electrophoresis Ch 30 Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substratesCh 31 The Synthesis and proteasomal degradation of a model substrate Ub5DHFR Ch 32 Assaying Degradation and Deubiquitination of a ubiquitinated substrate by purified 26S proteasomesCh 33 Probing the Ubiquitin/Proteasome System with Ornithine Decarboxylase, a Ubiquitin-Independent SubstrateCh 34 Atomic force microscopy of the proteasomeCh 35 Characterization of noncompetitive regulators of the proteasome activityCh 36 Development and use of anti-proteasome monoclonal antibodiesSection V - IsopeptidasesCh 37 Preparation and characterization of yeast and human desumoylating enzymesCh 38 Purification of the COP9 signalosome from porcine spleen, human cell lines and from Arabidopsis thaliana plants Ch 39 Purification method of the COP9 signalosome from human erythrocytesCh 40 UBP43, an ISG15-specific deconjugating enzyme: Expression, purification and enzymatic assaysCh 41 Strategies for assaying deubiquitinating enzymesCh 42 In vitro cleavage of Nedd8 from cullin 1 by COP9 Signalosome and Deneddylase 1Ch 43 Ubiquitin-ovomucoid fusion proteins as model substrates for monitoring degradation and deubiquitination by proteasomesCh 44 Using deubiquitinating enzymes as research toolsCh 45 Functional annotation of deubiquitinating eyzymes using RNA interference