Chemical synthesis of activity-based Diubiquitin probes

Abstract

Activity-based diubiquitin probes are highly useful in probing the deubiquitinase (DUB) activity and ubiquitin chain linkage specificity. Here we describe a detailed protocol to synthesize a new class of diubiquitin DUB probes. In this method, two ubiquitin moieties are connected through a linker resembling the native linkage in size and containing a Michael acceptor for trapping the DUB active-site cysteine. Detailed procedures for generating the linker molecule are also described.