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Applications: Study enzyme kinetics and screening small molecule inhibitors for drug discovery and high-throughput screening (HTS) applications.
Background: Ubiquitin specific peptidase 10 (USP10), 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 and ubiquitin isopeptidases. They contribute to the ubiquitin signaling pathway by countering the signal induced by ubiquitin conjugases and ligases. USP10 is a cysteine protease involved in ubiquitin recycling, DNA damage response, stress granule formation and can function as a tumor suppressor or oncogene. Some of its targets include p53, CFTR (cystic fibrosis transmembrane conductance regulator), SIRT6 (sirtuin 6) and NEMO (nuclear factor kappa B-essential modulator). UPS10 plays a role in cancer development, neurodegenerative diseases such as Alzheimeirʹs disease, cystic fibrosis and infections. The development of inhibitors for USP10 may constitute a promising avenue in the treatment of USP10 linked pathogenesis.
Description: The USP10 Inhibitor Screening Assay Kit is a fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP10 (Ubiquitin Specific Peptidase 10) for screening and profiling applications. The kit comes in a convenient 96-well format and contains enough purified recombinant human USP10 protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions.To determine the effect of an inhibitor on USP10 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 (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 and fluorescence with λexcitation/λemission maxima of 350/460 nm is emitted. 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.
Uniprot: Q14694
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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