A fluorescent in vitro assay to investigate paralog-specific SUMO conjugation

Nathalie Eisenhardt, Viduth K. Chaugule, Andrea Pichler (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

2 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationSUMO
Subtitle of host publicationmethods and protocols
EditorsManuel S. Rodriguez
Place of PublicationNew York
PublisherSpringer
Pages67-78
Number of pages12
Volume1475
ISBN (Electronic)9781493963584
ISBN (Print)9781493963560
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in molecular biology
PublisherSpringer
Volume1475
ISSN (Print)1064-3745

Keywords

  • SUMO
  • SUMO chains
  • E3 ligase
  • RanBP2
  • PIAS1
  • ZNF451
  • SUMO protease
  • In vitro sumoylation
  • Sp100

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  • Cite this

    Eisenhardt, N., Chaugule, V. K., & Pichler, A. (2016). A fluorescent in vitro assay to investigate paralog-specific SUMO conjugation. In M. S. Rodriguez (Ed.), SUMO: methods and protocols (Vol. 1475, pp. 67-78). (Methods in molecular biology; Vol. 1475). Springer . https://doi.org/10.1007/978-1-4939-6358-4_5