Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function is a vital first step to uncovering their cellular roles. Here, we provide protocols for the rapid analysis of DUB activity by activity-based labelling with the suicide probe, HA-ubiquitin vinyl sulfone (HA-UbVS), and ubiquitin chain disassembly assays. We have previously used these methods to analyse the activity of the DUB, UBP6, but in principle, these protocols are applicable to any DUB of interest.