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Applications: Enzyme kinetics and screening small molecule inhibitors for drug discovery and high-throughput applications.
Background: Ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3, also known as Ubiquitin thioesterase L3), belongs to a large group of ubiquitin-specific proteases capable of cleaving ubiquitin from other proteins. These enzymes are also referred to as deubiquitinating peptidases, deubiquitinases (DUBs), ubiquitin proteases, ubiquitin hydrolases or ubiquitin isopeptidases. They remove the covalently bound ubiquitin and contribute to the ubiquitin signaling pathway by countering the signal induced by ubiquitin conjugating enzymes and ligases. DUBs are a new therapeutic target for neurodegenerative diseases, cancer, diabetes, and autoimmune pathologies.
Description: The UCHL3 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme UCHL3 for screening and profiling applications. To determine the effect of an inhibitor on UCHL3 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.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 λexcitation/λemission maxima of 350/460 nm. The increase in fluorescence is proportional to the DUB activity.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Supplied As: The kit contains enough purified UCHL3 protein, Ubiquitinated-AMC substrate and assay buffer for 100 reactions.
Uniprot: P15374
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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