USP8 Inhibitor Screening Assay Kit
The USP8 Inhibitor Screening Assay Kit is a fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP8 (ubiquitin specific peptidase 8) for screening and profiling applications. The kit comes in a convenient 96-well format and contains enough purified recombinant human USP8 protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions.
To determine the effect of an inhibitor on USP8 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.
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 the fluorochrome AMC is quenched. Upon release from Ub, AMC emissions are no longer quenched fluorescence with λexcitation/λemission maxima of 350/460 nm is emitted. The increase in fluorescence is proportional to the DUB activity.
Need us to run inhibitor screens or profile your compounds against USP8? Check out our Protease Screening Services or Ubiquitination Screening Services.
- Adjustable micropipettor and sterile tips
- Plate reader
| Catalog # | Name | Amount | Storage |
| 80358 | USP8 FLAG-Tag* | 3 µg | -80°C |
| 81150 | Ub-AMC Substrate | 5 µl | -80°C |
| 79274 | 10x PR-01 Assay Buffer | 3 x 1 ml | -80°C |
| 0.5 M DTT | 200 µl | -80°C | |
| 79685 | 96-well black microplate | 1 | Room Temp |
* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.
Ubiquitin specific peptidase 8 (USP8), 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, ubiquitin isopeptidases. They contribute to the ubiquitin signaling pathway by countering the signal induced by ubiquitin conjugases and ligases. USP8 is involved in protein endosomal sorting and has been linked to several cancer types (lung, cervical, breast cancer and hepatocellular carcinoma). USP8 promotes the development of drug resistance in prostate cancer (PCa), with knockdown of USP8 resulting in docetaxel being more effective. In addition, its knockdown it suppresses EGFR (epidermal growth factor receptor), PI3K (phosphoinositide 3-kinase) and NF-κB (nuclear factor kappa-light chain enhancer of activated B cells) pathways. Further studies into the function of USP8 and the discovery of new inhibitors will open avenues of treatment for diseases like PCa.
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