The USP8 Inhibitor Screening Assay Kit is a fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP8 (ubiquitin specific peptidase 8) for screening and profiling applications. The kit comes in a convenient 96-well format and contains enough purified recombinant human USP8 protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions.

To determine the effect of an inhibitor on USP8 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 (Ub-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 the fluorochrome AMC is quenched. Upon release from Ub, AMC emissions are no longer quenched fluorescence with λexcitation/λemission maxima of 350/460 nm is emitted. The increase in fluorescence is proportional to the DUB activity.