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Single-Turnover RING/U-Box E3-Mediated Lysine Discharge Assays

  • Lori Buetow
  • Mads Gabrielsen
  • Danny T. HuangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)

Abstract

RING and U-box ubiquitin ligases promote ubiquitin (Ub) transfer by priming Ub-conjugated E2 in a closed conformation to optimize the thioester bond for nucleophilic attack by substrate lysine. Here, we describe a single-turnover lysine discharge assay for direct assessment of the activity of any RING/U-box E3-E2~Ub complex.

Key words

Ubiquitination Lysine discharge RING or U-box E3 ligases Radiolabeling 

Notes

Acknowledgments

This work was supported by Cancer Research UK (A23278) and European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement n° 647849).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Lori Buetow
    • 1
    • 2
  • Mads Gabrielsen
    • 1
    • 2
  • Danny T. Huang
    • 1
    • 2
    Email author
  1. 1.Cancer Research UK Beatson InstituteGlasgowUK
  2. 2.Institute of Cancer SciencesUniversity of GlasgowGlasgowUK

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