A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation

Eisenhardt, Nathalie; Chaugule, Vidith; Pichler, Andrea

doi:10.1007/978-1-4939-6358-4_5

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A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation

MPS-Authors

Eisenhardt, Nathalie
Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

Chaugule, Vidith
Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Pichler, Andrea
Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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https://link.springer.com/protocol/10.1007%2F978-1-4939-6358-4_5
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Citation

Eisenhardt, N., Chaugule, V., & Pichler, A. (2016). A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation. In Methods in Molecular Biology (pp. 67-78). New York: Springer + Business.

Cite as: https://hdl.handle.net/11858/00-001M-0000-002C-AF45-7

Abstract

Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.