USP9X Inhibitor Screening Assay Kit

The USP9X Inhibitor Screening Assay Kit is a fluorogenic assay kit designed to measure the activity of the deubiquitinating (DUB) enzyme USP9X (ubiquitin specific peptidase 9 X-linked) for screenin82726-1g and profiling applications. The kit contains enough purified USP9X, Ubiquitinated-AMC substrate, and assay buffer for 96 reactions. To determine the effect of an inhibitor on USP9X 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. Figure 1: Illustration of the assay principle. Ubiquitin-AMC is a fluorogenic substrate for ubiquitin hydrolases, with a 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.