Titlebook: Deubiquitinases; Methods and Protocol Julie Maupin-Furlow,Mariola J. Edelmann Book 2023 The Editor(s) (if applicable) and The Author(s), un

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Kathleen Stewart Hornsby,Kraig Kingtrate hydrolysis by DUBs releases free Ub or polyubiquitin (polyUb) chains. Whereas most quantitative DUB assays depend on fluorescently labeled artificial substrates, employing a sensor able to detect Ub release in real time makes it possible to monitor DUB activity using virtually any Ub conjugate

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Continuous Spatiotemporal Trajectory Joinsterization in vitro has been challenging. Here we describe an in vitro assay to characterize the biochemical activity of full-length Nsp3 isolated from cells. The assay can be used to evaluate Nsp3 inhibitors.

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Francesco Dramis,Giandomenico Fubelli probes, with immunoblotting and mass spectrometry proteomics-based readouts. Different types of activity-based protein profiling (ABPP) for studying the potency and selectivity of DUB inhibitors are outlined here, including the standard ABPP, the deep DUBome ABPP, and the ABPP-HT (high-throughput compatible).

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Synthetic Diubiquitin Fluorogenic Substrates to Study DUBs,n 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.

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Fluorometric Characterization of DUB Activity: From Single-Enzyme Reactions to Live Intact Cells, analysis of DUB activity. DUB activity can be measured in purified enzyme reactions, in cell lysates, or in intact cells depending upon the choice of the fluorometric probe. This chapter describes protocols and potential analysis tools to investigate DUB activity in these three scenarios.