SUMO protocols / edited by Helle D. Ulrich. — New York : Humana Press, c2009. – (58.17/M592/v.497)

Contents

    CONTENTS
    
    Preface
    Contributors
    PART I: INTRODUCTION
    1. The SUMO System: An Overview
    PART II: IDENTIFICATION OF SUMO TARGETS
    2. Identification of SUMO Target Proteins by Quantitative Proteomics
    3. Identification of SUMO-Conjugated Proteins and their SUMO Attachment Sites Using Proteomic Mass Spectrometry
    4. Identification of SUMO Targets Through In Vitro Expression Cloning
    5. Enhanced Detection of In Vivo SUMO Conjugation by Ubc9 Fusion-Dependent Sumoylation (UFDS)
    6. In Vivo Detection and Characterization of Sumoylation Targets in Saccharomyces cerevisiae
    PART III: IDENTIFICATION OF SUMO INTERACTORS
    7. Identification of SUMO-Interacting Proteins by Yeast Two-Hybrid Analysis
    8. Identification of SUMO-Binding Motifs by NMR
    PART IV: FUNCTIONAL ANALYSIS OF SUMOYLATION IN VIVO
    9. Regulation of Transcription Factor Activity by SUMO Modification 141
    10. Characterization of the Effects and Functions of Sumoylation Through Rapamycin-Mediated Heterodimerization
    PART V: BIOCHEMICAL STUDIES OF SUMO CONJUGATION
    11. Purification of SUMO Conjugating Enzymes and Kinetic Analysis of Substrate Conjugation
    12. Performing In Vitro Sumoylation Reactions Using Recombinant Enzymes
    13. Preparation of Sumoylated Substrates for Biochemical Analysis
    14. Strategies for the Expression of SUMO-Modified Target Proteins in Escherichia coli
    PART VI: BIOCHEMICAL STUDIES OF SUMO DECONJUGATION
    15. Preparation of SUMO Proteases and Kinetic Analysis Using Endogenous Substrates
    16. An In Vitro FRET-Based Assay for the Analysis of SUMO Conjugation and Isopeptidase Cleavage
    17. FRET-Based In Vitro Assays for the Analysis of SUMO Protease Activities
    18. Detection and Characterization of SUMO Protease Activity Using a Sensitive Enzyme-Based Reporter Assay 269
    PART VII: MANIPULATION AND ALTERNATIVE USES OF THE SUMO SYSTEM
    19. Inhibition of the SUMO Pathway by Gain1 285
    20. SUMO Fusion Technology for Enhanced Protein Production in Prokaryotic and Eukaryotic Expression Systems 303