USP5 Inhibitor Screening Assay Kit

• Background:

  Ubiquitin specific peptidase 5 (USP5) 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 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 USP5 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP5 for screening and profiling applications. The kit contains enough purified USP5 protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions.To determine the effect of an inhibitor on USP5 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 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 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 proteolysis, AMC is no longer quenched and emits fluorescence with an λexcitation/λemission 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 USP5? Check out ourProtease Screening ServicesorUbiquitination Screening Services.

• UniProt:

  P45974

• Applications:

  Enzyme kinetics studies and screening small molecule inhibitors for drug discovery and high-throughput screen applications.

• Format:

  Catalog #NameAmountStorage80355USP5 (IsoT), FLAG-Tag*>1 µg-80°C81150Ub-AMC Substrate5 µl-80°C7927410x PR-01 Assay Buffer3 x 1 ml-80°C0.5 M DTT200 µl-20°C7968596-well black microplate1Room Temp* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.

• Shipping Conditions:

  -80°C

• Storage Conditions:

  This assay kit will perform optimally for up to 6 monthsfrom date of receipt when the materials are stored as directed.

• Notes:

  Troubleshooting GuideVisitassay-kits-faqfor detailed troubleshooting instructions. For all further questions, please email[email protected]

• Contraindications:

  The final concentration of DMSO in the assay should not exceed 1%.Fluorescent compounds that have λex=350 nm and detection at λem=460 nm can interfere with the readouts.The presence of strong acids or bases, ionic detergents and high salt should be avoided.