This volume covers methods to study protein modification by the Small Ubiquitin-like Modifier (SUMO) family of proteins, post-translational modifiers with essential roles in physiology and pathology. The book explores methods to detect SUMOylated proteins, identify SUMO modified sites, characterize the deposition and removal of these modifications, and explore how SUMO modifies protein-protein interactions both in vitro and in cells. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips for troubleshooting and avoiding known pitfalls.
Authoritative and practical, SUMO: Methods and Protocols serves as an ideal guide for researchers seeking the most relevant and cutting-edge technologies to dissect protein SUMOylation.
The SUMO Pathway.- SUMO Antibody Validation.- Native and Endogenous SUMO Site Identification Using Mass Spectrometry (NESSI-MS).- A Fluorometric Assay to Compare SUMO Isoforms Preferences in the SENP/ULP Family.- Production and Purification of SUMO-UBC9 and SUMO-RANGAP1CTD.- Synthesizing Defined Ubiquitin-Modified SUMO Dimers.- In Vitro SUMOylation Assays with a DNA-Sensitive Multi-Subunit E3 Ligase.- Cell-Based Assays to Study SUMOylation in Drosophila.- SUMO-Activated Target Traps (SATTs) for the Identification of Specific SUMOylated Proteins for an E3 Ligase of Interest.- Synthesis and Application of SUMO-Based Probes for SUMO Protease Profiling.- Measurement of Substrate SUMOylation in Real Time by Fluorescence Spectroscopy.- Production and Purification of Recombinant SUMOylation Enzymes and SUMO Proteins.- Non-Hydrolysable ADP-Ribosylated SUMO2 via Copper(I) Catalyzed Azide Alkyne Cycloaddition.- A Vivo-Morpholino-Based Approach to Block Paralog-Specific SUMOylation.- Bimolecular Fluorescence Complementation Assay to Evaluate the Interaction between SUMO and Its Target Proteins in Cells.
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