Synthetic Diubiquitin Fluorogenic Substrates to Study DUBs

Abstract

Development of (semi-)synthetic methods to prepare ubiquitin (Ub)-based reagents has proven to be helpful in the classification of deubiquitinating proteases (DUBs). To study DUB selectivity for one or more of the eight possible poly-Ub chains, fluorogenic assay reagents have been reported relying on the appearance of a fluorescent signal upon DUB-mediated cleavage of the reagent. In this protocol, we describe the use of such an assay to profile the selectivity of TRABID, a member of the OTU family of DUBs.

Keywords: Activity assay; Deubiquitinating protease; Fluorescence; Fluorogenic substrate; TRABID.