Deubiquitinating Enzyme (DUBs) Activity Assay Kits

• Utilizes fluorescent substrates for detection
• Quickly screen for DUB inhibitors
• Sensitive and specific
• Use for kinetic or endpoint analysis
• Easily detected using a microplate reader for an excitation wavelength of 485 nm and an emission wavelength of 535 nm

Ubiquitination and deubiquitination are two important physiological processes in the ubiquitin-proteasome system, responsible for protein degradation in cells. The deubiquitinating enzyme (DUB) family contains ~100 proteins that remove ubiquitin from a variety of substrates. Deubiquitinating enzymes (DUBs) are widely involved in the regulation of various cellular processes, such as cell proliferation, autophagy, DNA damage repair, and immune response. They play important roles in the development of malignant tumors, neurodegenerative diseases, and other immune-related diseases. In addition, some bacteria and viruses, such as SARS-CoV, also express pathogenic DUBs to suppress host immune responses and increase their chances of survival and replication. Hence, DUBs represent novel candidates for target-directed drug development.

Ubiquitin derivatives conjugated with various fluorophores have been reported as substrates for biochemical DUB assays. Ubiquitin-Rhodamine 110 (Ub-Rho110) is a fluorogenic rhodamine-based substrate. Ub-Rho110 is an ubiquitin substrate whose C-terminal derivatives are Rho110. While rhodamine is in the Ub-Rho110 moiety, it is di-substituted thereby quenching the intrinsic fluorescence. However, mono-substituted rhodamine, which exhibits intense fluorescence, is released by the DUBs, thus allowing the real-time monitoring of the sensitivity or activity of DUBs by monitoring the 535 nm emission wavelengths after using 485 nm excitation. With these longer wavelengths,the risks of artifacts in auto-fluorescence are reduced. The increase in fluorescence is proportional to the DUB activity.

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