Observing Real-Time Ubiquitination in High Throughput with Fluorescence Polarization

Abstract

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.