The DUB-Freedom™ Inhibitor Screening Assay Kit is a fluorogenic assay designed to measure the activity of several purified deubiquitinating (DUB) enzymes of interest in a homogeneous 96-reaction format and is ideal for screening and profiling applications. The kit contains enough Ubiquitinated-AMC substrate and assay buffer for 100 reactions. It also includes the purified catalytic domain of the DUB enzyme USP2 (ubiquitin carboxyl-terminal hydrolase 2) (amino acids 259-end) as a positive control and Ub-Aldehyde as control inhibitor.

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 the purified catalytic domain of human USP2 protein and 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 (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 AMC is quenched. Upon release from Ub, AMC emissions are no longer quenched and fluorescence with λexcitation/λemission maxima of 350/460 nm is emitted. The increase in fluorescence is proportional to the DUB activity.