Observing Real-Time Ubiquitination in High Throughput with Fluorescence Polarization

Tyler G. Franklin, Jonathan N. Pruneda

Research output: Chapter in Book/Report/Conference proceedingChapter


Reconstitution of ubiquitin conjugation and deconjugation in vitro provides access to valuable information on enzyme kinetics, specificity, and structure–function relationships. Classically, these biochemical assays culminate in separation by SDS-PAGE and analysis by immunoblotting, an approach that requires additional time, can be difficult to quantify, and provides granular snapshots of the reaction progression. To address these limitations, we have implemented a fluorescence polarization-based assay that tracks ubiquitin conjugation and deconjugation in real time based upon changes in molecular weight. We find this approach, which we have termed “UbiReal,” to greatly facilitate biochemical studies such as mutational analyses, specificity determination, and inhibitor characterization.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages10
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Deubiquitinase
  • E3 ligase
  • Fluorescence polarization
  • High throughput
  • Inhibitor
  • Ubiquitin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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