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Application: Enzyme kinetics studies and screening small molecule inhibitors for drug discovery and high-throughput screening (HTS) applications.
Background: USP13 (ubiquitin carboxyl-terminal hydrolase 13), also known as isopeptidase T 3 (ISOT-3) is a DUB (deubiquitination) enzyme. It participates in multiple crucial cellular processes, such as autophagy, DNA damage response (DDR), and ERAD (endoplasmic reticulum-associated degradation) by deubiquitinating proteins involved in those functions. It also plays a role in diseases such as cancer and neurodegeneration. It can play both a suppressor and activator role in tumorigenesis. For instance, low levels of USP13 correlate with low levels of PTEN (phosphatase and tensin homolog), as UPS13 stabilizes the protein. The development of USP13 inhibitors has been of interest, however due to the similarity between USP13 and USP10 can be hard to accomplish. A deeper understanding of all its cellular roles and how to best target its function in a specific context may prove beneficial in cancer therapy.
Contraindications: The final concentration of DMSO in the assay should not exceed 1%.Compounds that are fluorescent may interfere with the results, depending on their spectral excitation and emission properties.It is recommended that the compound alone is tested to determine any potential interference of the compound with the assay results.
Description: The USP13 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP13 (ubiquitin specific peptidase 13) for screening and profiling applications. The kit contains enough purified USP13 protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions. To determine the effect of an inhibitor on USP13 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 proteases (MJD). 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 fluorochrome AMC is quenched. Upon proteolysis, AMC is no longer quenched and emits fluorescence with excitation/emission maxima of 350/460 nm. The increase in fluorescence is proportional to the DUB activity. Need us to run inhibitor screens or profile your compounds against USP13? Check out our Protease Screening Services or Ubiquitination Screening Services.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Target: USP13
Uniprot: Q92995
Unit Definition: Assay Kit
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: Liao X., et al., 2022 Front. Cell Dev. Biol. 10:3389.
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