The Ubiquitin specific peptidase 14 (USP14) Inhibitor Screening Assay Kit is a 96-well fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP14 for screening and profiling applications. The kit contains enough purified USP14 protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions.

To determine the effect of an inhibitor on USP14 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 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 based on the C-terminus derivatization of ubiquitin with 7-amido-4-methylcoumarin (AMC). In conjugated form, the energy emitted from the fluorochrome AMC is quenched. Upon proteolysis, AMC is no longer quenched and emits fluorescence with λexcitation/λemission maxima of 350/460 nm. The increase in fluorescence is proportional to the DUB activity.