Ubiquitin Proteasome Protocols 1st Edition by Cam Patterson, Douglas M Cyr ISBN 1588292525 9781588292520
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ISBN 10: 1588292525
ISBN 13: 9781588292520
Author: Cam Patterson, Douglas M Cyr
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.
Ubiquitin Proteasome Protocols 1st Table of contents:
Chapter 1: Introduction to the Ubiquitin-Proteasome System
1.1 Overview of Protein Degradation
1.2 Ubiquitination: Mechanism and Enzymes
1.3 The 26S Proteasome: Structure and Function
1.4 Biological Significance and Disease Implications
Chapter 2: Reagents, Buffers, and General Considerations
2.1 Common Reagents for Ubiquitination and Proteasome Assays
2.2 Preparation of Buffers and Solutions
2.3 Sample Handling and Storage
2.4 Safety and Troubleshooting
Chapter 3: In Vitro Ubiquitination Assays
3.1 E1, E2, E3 Enzyme Reactions
3.2 Substrate Preparation
3.3 Detection of Ubiquitinated Proteins
3.4 Quantification Methods
Chapter 4: Proteasome Activity Assays
4.1 Fluorogenic Substrate Assays
4.2 ATP-Dependent vs. ATP-Independent Assays
4.3 Native Gel and In-Gel Activity Assays
4.4 Inhibitor Studies
Chapter 5: Immunoprecipitation and Western Blotting of Ubiquitinated Proteins
5.1 Sample Preparation
5.2 Immunoprecipitation Protocols
5.3 Detection by Western Blot
5.4 Troubleshooting
Chapter 6: Cell-Based Ubiquitin-Proteasome Assays
6.1 Transient and Stable Expression of Tagged Proteins
6.2 Proteasome Inhibition Studies
6.3 Pulse-Chase Experiments
6.4 Monitoring Protein Turnover in Cells
Chapter 7: Advanced Techniques
7.1 Mass Spectrometry for Ubiquitin Site Mapping
7.2 Quantitative Proteomics of Proteasome Substrates
7.3 CRISPR/Cas Approaches to Study Ubiquitination
7.4 High-Throughput Screening for Proteasome Modulators
Chapter 8: Troubleshooting and Optimization
8.1 Common Pitfalls and How to Avoid Them
8.2 Tips for Enhancing Sensitivity and Specificity
8.3 Data Analysis and Interpretation
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