Description
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
Biochemical Methods to Study the Ubiquitin-Proteasome System.- Small-Molecule Inhibitors of Proteasome Activity.- Purification of E1 and E1-Like Enzymes.- Assays for RING Family Ubiquitin Ligases.- Ubiquitin Chain Synthesis.- Purification of Proteasomes, Proteasome Subcomplexes, and Proteasome-Associated Proteins From Budding Yeast.- Recognition and Processing of Misfolded Proteins by PA700, the 19S Regulatory Complex of the 26S Proteasome.- Cell-Free Assay for Ubiquitin-Independent Proteasomal Protein Degradation.- Assays of Proteasome-Dependent Cleavage Products.- Identification of Components of Protein Complexes.- Mass Spectrometric Determination of Protein Ubiquitination.- Reconstitution of Endoplasmic Reticulum-Associated Degradation Using Yeast Membranes and Cytosol.- Reticulocyte Lysate as a Model System to Study Endoplasmic Reticulum Membrane Protein Degradation.- Deubiquitinating Enzyme Purification, Assay Inhibitors, and Characterization.- Cellular Methods to Study Ubiquitin-Proteasome-Dependent Functions.- Measuring Ubiquitin Conjugation in Cells.- Assays for Proteasome Assembly and Maturation.- N-Terminal Ubiquitination.- Quantitating Defective Ribosome Products.- Endoplasmic Reticulum-Associated Protein Quality Control and Degradation.- Endoplasmic Reticulum-Associated Protein Quality Control and Degradation.- Cystic Fibrosis Transmembrane Conductance Regulator as a Model Substrate to Study Endoplasmic Reticulum Protein Quality Control in Mammalian Cells.- Aggresome Formation.- Detection of Sumoylated Proteins.- Proteasome Inhibitors in Cancer Therapy.- Parkinson’s Disease.
