Category: Assay Development and Screening

1294-E - Development of a Real-Time Homogeneous TR-FRET Ubiquitin Conjugation and Deconjugation Assay Platform

Wednesday, February 7, 2018
11:30 AM - 12:30 PM

South Bay Bio LLC

 Todays preferred choice of drug screening technology is TR-FRET, which uses the extended fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a short time delay between FRET donor excitation and emission. This delay provides a means to separate “true” signal from short-lived background fluorescence, and reduce interference from compound fluorescence and other assay artifacts. Here, we report the performance and capabilities of a new homogeneous real-time TR-FRET based assay platform developed for the ubiquitin proteasome system. Using ubiquitin either labeled with Europium-Cryptate (donor) or Cyanine5 (acceptor), we demonstrate for the first time ubiquitin conjugation and deconjugation measured homogeneously in Real-Time, with assays commonly exhibiting Z’ ≥ 0.8. We show examples of auto-ubiquitination kinetics of several human recombinant E3 ligases of significant interest, namely MDM2, NEDD4, ITCH, Parkin (wt and W403A), and XIAP.  In addition, we also show TR-FRET based deconjugation kinetics of auto-ubiquitinated ligases using USP2cd in comparison to other commercial substrates.  Furthermore, assays shown have been optimized for low volume 384-well plates making them well suited for HTS. Coupled with the assays’ short development time (as no antibody development is required for assay optimization), the platform is ideally suited for a wide variety of academic and industry screening applications.

Anthony Mauriello

Managing Director
South Bay Bio
San Jose, CA

Founder of South Bay Bio LLC.