The Ubiquitin Proteasome System

Methods and Protocols

Thibault Mayor, Gary Kleiger (Herausgeber)

Buch | Hardcover

413 Seiten

2018 | 1st ed. 2018
Humana Press Inc. (Verlag)
978-1-4939-8705-4 (ISBN)

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“This volume explores numerous techniques used to study the ubiquitin proteasome system. The chapters in this book are organized into five parts and cover topics such as determining the mechanisms of action for E2s, E3s, and DUB enzymes; the latest advances to study the formation of poly-ubiquitin chains as well as their linkage types; the binding partners of proteins in the UPS; methods for structure determination by x-ray crystallography, cryo electron microscopy and SAXS; screening assays to select for degrons or modulators of E3s and DUBs; proteomics approaches in the ubiquitin field and methods to study 26S proteasome function. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Thorough and authoritative, The UbiquitinProteasome System: Methods and Protocols is a valuable resource for both experienced and novice scientists who are interested in expanding their knowledge in this field.

Characterization of RING-Between-RING E3 Ubiquitin Transfer Mechanisms.- Single Turnover RING/U-Box E3-Mediated Lysine Discharge Assays.- Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins.- Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for their Ubiquitin Ligase Partners.- Competition Assay for Measuring Deubiquitinating Enzyme Substrate Affinity.- Enzymatic Assembly of Ubiquitin Chains.- Ubiquitin-Activated Interaction Traps (UBAITs): Tools for Capturing Protein-Protein Interactions.- Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries.- Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells.- A Method to Monitor Protein Turnover by Flow Cytometry and to Screen for Factors that Control Degradation by Fluorescence-Activated Cell Sorting.- E. coli Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins.- Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates during E3-Mediated Ubiquitin-Like Protein Ligation.- Small-Angle X-Ray Scattering for the Study of Proteins in the Ubiquitin Pathway.- Methods for Preparing Cryo-EM Grids of Large Macromolecular Complexes.- Recombinant Expression, Unnatural Amino-Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes.- Native Gel Approaches in Studying Proteasome Assembly and Chaperones.- Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways.- Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis.- Measurement of the Multiple Activities of 26S Proteasomes.- Exploring the Regulation of Proteasome Function by Subunit Phosphorylation.- Scalable In Vitro Proteasome Activity Assay.- Exploring the Rampant Expansion of Ubiquitin Proteomics.- Ubiquitin diGLY Proteomics as an Approach to Identify and Quantify the Ubiquitin-Modified Proteome.- Interpreting the Language of Polyubiquitin with Linkage-Specific Antibodies and Mass Spectrometry.- Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking Assisted Affinity Purification and Mass Spectrometry.

Erscheinungsdatum	01.10.2018
Reihe/Serie	Methods in Molecular Biology ; 1844
Zusatzinfo	39 Illustrations, color; 29 Illustrations, black and white; XV, 413 p. 68 illus., 39 illus. in color.
Verlagsort	Totowa, NJ
Sprache	englisch
Maße	178 x 254 mm
Themenwelt	Naturwissenschaften ► Biologie ► Biochemie
Themenwelt	Naturwissenschaften ► Biologie ► Mikrobiologie / Immunologie
Schlagworte	Cul4 • E2 enzymes • flow cytometry • poly-ubiquitin chains • Ubiquitination
ISBN-10	1-4939-8705-4 / 1493987054
ISBN-13	978-1-4939-8705-4 / 9781493987054
Zustand	Neuware