DUB-Freedom™ Inhibitor Screening Assay Kit
The DUB-Freedom™ Inhibitor Screening Assay Kit is a fluorogenic assay designed to measure the activity of several purified deubiquitinating (DUB) enzymes of interest in a homogeneous 96-reaction format and is ideal for screening and profiling applications. The kit contains enough Ubiquitinated-AMC substrate and assay buffer for 100 reactions. It also includes the purified catalytic domain of the DUB enzyme USP2 (ubiquitin carboxyl-terminal hydrolase 2) (amino acids 259-end) as a positive control and Ub-Aldehyde as control inhibitor.
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 the purified catalytic domain of human USP2 protein and 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 (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 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.
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- Purified DUB of interest
- Adjustable micropipettor and sterile tips
- Plate reader
| Catalog # | Name | Amount | Storage |
| 80392 | USP2, His-Tag (E. coli-derived)* | > 1 µg | -80°C |
| 81150 | Ub-AMC Substrate | 5 µl | -80°C |
| 79274 | 10x PR-01 Assay Buffer | 3 x 1 ml | -80°C |
| Ub-Aldehyde (350 µM) | 5 µl | -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.
Deubiquitinases (DUBs), also known as deubiquitinating peptidases, ubiquitin proteases, ubiquitin hydrolases, ubiquitin isopeptidases, are enzymes that remove ubiquitin or ubiquitin chains from other proteins. These proteins contribute to the ubiquitin signaling pathway by countering the signal induced by ubiquitin ligases and thereby control the stability, subcellular localization and/or interactions of many cellular proteins. DUBs are implicated in many human pathologies including neurodegenerative diseases, cancer, diabetes, and autoimmune pathologies, making them interesting new therapeutic targets.
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