The USP1 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP1 for screening and profiling applications. To determine the effect of an inhibitor on USP1 activity, the enzyme should be preincubated with or without the test inhibitor prior to adding the Ub-AMC substrate to the reaction. The assay was functionally validated using Ub-Aldehyde, a potent inhibitor of the DUB subfamilies Ubiquitin C-terminal Hydrolases (UCHs), Ubiquitin-Specific Proteases (USPs), Ovarian Tumor Proteases (OTU), and Machado-Josephin Domain (MJD) proteases. Ubiquitin-AMC is a fluorogenic substrate for ubiquitin hydrolases based on the C-terminus derivatization of ubiquitin with 7-amido-4-methylcoumarin (AMC). In the conjugated form, the energy emitted from fluorochrome AMC is quenched. Upon proteolysis, AMC is no longer quenched and emits fluorescence with an lambdaexcitation/lambdaemission maxima of 350/460 nm. The increase in fluorescence is proportional to the DUB activity.
