Ubiquitin and Protein Degradation, Part A, Volume 398

Section I – E1 Enzymes Ch 1 High level expression and purification of recombinant E1 enzyme Ch 2 Expression, purification and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex Ch 3 A FRET-based assay to study SUMO modification in solution Ch 4 Properties of the ISG15 E1 enzyme UbE1L

Section II – E2 Enzyme Ch 5 Purification and properties of the Ubiquitin Conjugating Enzymes Cdc34 and Ubc13-Mms2 Ch 6 Eexpression, purification, and properties of Ubc4/5 family E2 enzymes Ch 7 In vitro systems for Nedd8 conjugation by Ubc12 Ch 8 Purification and activity assays for Ubc9, the ubiquitin conjugating enzyme for the small ubiquitin-like modifier SUMO Ch 9 High-througput assay to monitor Formation of the E2-Ubiquitin thioester intermediate

Section III – E3 Enzyme Ch 10 Expression and Evaluation of RING Finger proteins Ch 11 Expression and assay of HECT domain ligases Ch 12 High Level Expression and Purification of Recombinant SCF Ubiquitin Ligase Ch 13 In vitro reconstitution of SCF substrate ubiquitination with purified proteins Ch 14 Expression and assay of glycoprotein-specific ubiquitin ligases Ch 15 Affinity purification and assay of APC/C on p13 Suc1 Ch 16 Large-Scale Purification of the Vertebrate Anaphase-Promoting Complex/Cyclosome Ch 17 Purification and assay of the budding yeast Anaphase Promoting Complex Ch 18 Enzymology of the Anaphase-Promoting Complex Ch 19 Identification of Cell Cycle Dependent Phosphorylation Sites on the Anaphase-Promoting Complex/Cyclosome by Mass Spectrometry Ch 20 Purification and assay of Mad2: A Two-state Inhibitor of APC/C Ch 21 Identification, expression and assay of an oxidation-specific ubiquitin ligase, HOIL-1 Ch 22 (In revision)Purification and assay of chaperon-dependent ubiquitin ligase CHIP Ch 23 Methods for the functional genomic analysis of ubiquitin ligases

Section IV - Proteasome Ch 24 Purification of PA700, the 19S regulatory complex of the 26S Proteasome Ch 25 Purification and analysis of recombinant 11S acti vators of the 20S proteasome: Trypanosoma brucei PA26 and human PA28a, PA28B, and PA28y Ch 26 Purification and assay of proteasome activator PA200 Ch 27 Purification, crystallization and X-ray analysis of the yeast 20S proteasome Ch 28 (In revision due to figures only)Preparation of Hybrid (19S-20S-PA28) Proteasome Complexes and Analysis of Peptides Generated During Protein Degradation Ch 29 Characterization of the Proteasome using Native Gel Electrophoresis
Ch 30 Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates Ch 31 The Synthesis and proteasomal degradation of a model substrate Ub5DHFR
Ch 32 Assaying Degradation and Deubiquitination of a ubiquitinated substrate by purified 26S proteasomes Ch 33 Probing the Ubiquitin/Proteasome System with Ornithine Decarboxylase, a Ubiquitin-Independent Substrate Ch 34 Atomic force microscopy of the proteasome Ch 35 Characterization of noncompetitive regulators of the proteasome activity Ch 36 Development and use of anti-proteasome monoclonal antibodies

Section V - Isopeptidases Ch 37 Preparation and characterization of yeast and human desumoylating enzymes Ch 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 erythrocytes Ch 40 UBP43, an ISG15-specific deconjugating enzyme: Expression, purification and enzymatic assays Ch 41 Strategies for assaying deubiquitinating enzymes Ch 42 In vitro cleavage of Nedd8 from cullin 1 by COP9 Signalosome and Deneddylase 1 Ch 43 Ubiquitin-ovomucoid fusion proteins as model substrates for monitoring degradation and deubiquitination by proteasomes Ch 44 Using deubiquitinating enzymes as research tools Ch 45 Functional annotation of deubiquitinating eyzymes using RNA interference

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.

Topics include:
E1 Enzymes E2 Enzymes E3 Enzymes Proteasomes Isopeptidases

Biochemists, biophysicists, cell biologists, molecular biologists, geneticists, developmental biologists