Main description:

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.

Contents:

Part I Introduction

1. Concepts and Methodologies to Study Protein SUMOylation: An Overview
Michael J. Matunis and Manuel S. Rodriguez

2. The Regulation of Chromatin by Dynamic SUMO Modifications
Nicole R. Wilson and Mark Hochstrasser

Part II Procedures to Study Protein SUMOylation In Vitro

3. Reconstitution of the Recombinant RanBP1 SUMO E3 Ligase Complex
Tobias Ritterhoff, Hrishikesh Das, Yuqing Hao, Volkan Sakin, Annette Flotho, Andreas Werner, and Frauke Melchior

4. Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria

Frederique Brockly, Marc Piechaczyk, and Guillaume Bossis
5. A Fluorescent In Vitro Assay to Investigate Paralog Specific SUMO ConjugationNathalie Eisenhardt, Viduth K. Chaugule, and Andrea Pichler

6. Identification and Characterization of SUMO-SIM Interactions
Koraljka Husnjak, Jan Keiten-Schmitz, and Stefan Muller

7. Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions between SUMO-Traps and Mono or PolySUMO Moieties
Wendy Xolalpa, Manuel S. Rodriguez, and Patrick England

8. Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins
Valerie Lang, Elisa Da Silva-Ferrada, Rosa Barrio, James D. Sutherland, and Manuel S. Rodriguez

9. In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific ProteasesJulia Eckhoff and R. Jurgen Dohmen
Part III Analysis of Protein SUMOylation Using Cell Lines

10. Detection of Protein SUMOylation In Situ by Proximity Ligation AssaysUmut Sahin, Florence Jollivet, Caroline Berthier, Hugues de The, and Valerie Lallemand-Breitenbach<

11. In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized CellsEri Yuasa and Hisato Saitoh
12. Analysis of SUMOylated Proteins in Cells and In Vivo Using BioSUMO StrategyLucia Pirone, Wendy Xolalpa, Ugo Mayor, Rosa Barrio, and James D. Sutherland
13. Label-Free Identification and Quantification of SUMO Target ProteinsIvo A. Hendriks and Alfred C.O. Vertegaal
14. The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding ProteinsElisa Aguilar-Martinez and Andrew D. Sharrocks
15. Isolation of In Vivo SUMOylated Chromatin-Bound ProteinsTasneem Bawa-Khalfe16. Identification of Substrates of Protein-Group SUMOylationIvan Psakhye and Stefan Jentsch
17. Tools to Study SUMO Conjugation in Caenorhabditis elegansFederico Pelisch and Ronald T. Hay
18. Purification of SUMO Conjugates from Arabidopsis for Mass Spectrometry AnalysisTherese C. Rytz, Marcus J. Miller, and Richard D. Vierstra
19. Detection of SUMOylation in Plasmodium falciparumKatherine H. Reiter and Michael J. Matunis
20. Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1Marilyn Tirard and Nils Brose