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

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


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 



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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