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This volume explores various methodologies to study biochemical, molecular, and cellular biology aspects of some processes regulated by protein SUMOylation. SUMO: Methods and Protocols is organized into four parts, and starts with an historical overview on protein SUMOylation and a presentation of the methods included in the book. The first part also includes a review on chromatin regulation by dynamic SUMO modifications. The second part focuses on in vitro techniques, including biochemical methods to study mechanistic aspects of protein SUMOylation. The third part includes protocols to be used with cell cultures, which often are the first approaches used in most laboratories. The final part includes methodologies adapted for the analysis in vivo using distinct model organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include a brief introduction to the subject, a list of necessary materials and reagents, a step-by-step reproducible laboratory protocol ending with a Notes section on troubleshooting tips, and tips and strategies to avoid known pitfalls. Unique and cutting-edge, SUMO: Methods and Protocols provides a comprehensive source of protocols for specialists and researchers not familiar with this vital system.
Concepts and Methodologies to Study Protein SUMOylation: An Overview.- The Regulation of Chromatin by Dynamic SUMO Modifications.- Reconstitution of the Recombinant RanBP1 SUMO E3 Ligase Complex.- Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria.- A Fluorescent In Vitro Assay to Investigate Paralog Specific SUMO Conjugation.- Identification and Characterization of SUMO-SIM Interactions.- Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions between SUMO-Traps and Mono or PolySUMO Moieties.- Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins.- In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases.- Detection of Protein SUMOylation In Situ by Proximity Ligation Assays.- In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells.- Analysis of SUMOylated Proteins in Cells and In Vivo Using BioSUMO Strategy.- Label-Free Identification and Quantification of SUMO Target Proteins.- The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins.- Isolation of In Vivo SUMOylated Chromatin-Bound Proteins.- Identification of Substrates of Protein-Group SUMOylation.- Tools to Study SUMO Conjugation in Caenorhabditis elegans.- Purification of SUMO Conjugates from Arabidopsis for Mass Spectrometry Analysis.- Detection of SUMOylation in Plasmodium falciparum.- Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1.
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