1. Concepts and Methodologies to Study Protein SUMOylation: An Overview Michael J. Matunis and Manuel S. Rodríguez 2. The Regulation of Chromatin by Dynamic SUMO Modifications Nicole R. Wilson and Mark Hochstrasser Part IIProcedures 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
Frédérique Brockly, Marc Piechaczyk, and Guillaume Bossis
5. A Fluorescent In Vitro Assay to Investigate Paralog Specific SUMO Conjugation
Nathalie Eisenhardt, Viduth K. Chaugule, and Andrea Pichler
6. Identification and Characterization of SUMO-SIM Interactions
Koraljka Husnjak, Jan Keiten-Schmitz, and Stefan Müller
7. Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions between SUMO-Traps and Mono or PolySUMO Moieties
Wendy Xolalpa, Manuel S. Rodríguez, and Patrick England
8. Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins
Valérie Lang, Elisa Da Silva-Ferrada, Rosa Barrio, James D. Sutherland, and Manuel S. Rodríguez
9. In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases
Julia Eckhoff and R. Jürgen Dohmen
Part III Analysis of Protein SUMOylation Using Cell Lines
10. Detection of Protein SUMOylation In Situ by Proximity Ligation Assays
Umut Sahin, Florence Jollivet, Caroline Berthier, Hugues de Thé, and Valérie Lallemand-Breitenbach
<
11. In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells
Eri Yuasa and Hisato Saitoh
12. Analysis of SUMOylated Proteins in Cells and In Vivo Using BioSUMO Strategy
Lucia Pirone, Wendy Xolalpa, Ugo Mayor, Rosa Barrio, and James D. Sutherland
13. Label-Free Identification and Quantification of SUMO Target Proteins
Ivo A. Hendriks and Alfred C.O. Vertegaal
14. The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins
Elisa Aguilar-Martínez and Andrew D. Sharrocks
15. Isolation of In Vivo SUMOylated Chromatin-Bound Proteins
Tasneem Bawa-Khalfe
<
Part IV Procedures to Study Protein SUMOylation In Vivo<
16. Identification of Substrates of Protein-Group SUMOylation
Ivan Psakhye and Stefan Jentsch
17. Tools to Study SUMO Conjugation in Caenorhabditis elegans
Federico Pelisch and Ronald T. Hay
18. Purification of SUMO Conjugates from Arabidopsis for Mass Spectrometry Analysis
Thérèse C. Rytz, Marcus J. Miller, and Richard D. Vierstra
19. Detection of SUMOylation in Plasmodium falciparum
Katherine H. Reiter and Michael J. Matunis
20. Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1
Marilyn Tirard and Nils Brose
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.