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A natural substrate, ubiquitin, coupled with the AMC (7-amino-4-methylcoumarin) fluorophore is used in the kit. Upon cleavage by a deubiquitin protease, this substrate generates a blue fluorophore that can be detected at excitation/emission=354/442 nm. The substrate supplied in the kit is exquisitely sensitive for ubiquitin hydrolases (UCHs). The detection limit can reach as low as 3.7 pg/mL.
Modification of proteins with ubiquitin is a key step in protein degradation controlling many intracellular processes such as transcription, cell cycle progression, receptor internalization, and DNA repair.1 Recent studies have demonstrated that regulation also occurs at the level of deubiquitination.2,3 Deubiquitinating enzymes (DUBs) are proteases that reverse ubiquitin modifications.
Over 100 DUBs have been identified in humans. They are grouped into five distinct families based on their sequence similarities and mechanisms of action. Four of the families are cysteine proteases, while the fifth is a novel type of zinc-dependent metalloprotease.4,5 The majority of human DUBs belong to two cysteine proteases families, the ubiquitin specific proteases (USPs) and the ubiquitin carboxyterminal hydrolases (UCHs). Both protease families hydrolyze the peptide bond downstream of the C terminus of ubiquitin, which is either a classic peptide bond in the proforms of ubiquitin or an isopeptide bond to an e-amino group of a lysine residue within an ubiquitin modified protein. DUBs have been implicated in the pathogenesis of many human diseases, such as neurodegenerative disorders and cancer.6,7 Consequently they have become actively studied molecular targets for drug discovery.8,9
AnaSpec, EGT Group, is also pleased to introduce the SensoLyte® 520 Deubiquitination Assay Kit. This kit, which employs a peptide substrate conjugated to a green fluorophore, is also good for inhibitors/activators screening in deubiquitinylation or for continuous assay of enzyme activity. Bright green fluorescence is generated upon protease cleavage and can be detected at excitation/emission=490/520 nm. The long wavelength spectra of the green fluorophore released provides less interference from other reaction components.
References
1. Hershko, A. and Ciechanover, A. Annu Rev Biochem 67, 425 (1998).
2. Amerik, AY. and M. Hochstrasser, Biochim Biophys Acta 1695, 189 (2004)
3. Kim, J.H. et al. J Biochem. 134, 9 (2003).
4. Soboleva, T.A. Curr Protein Pepy Sci 5, 191 (2004).
5. Karen, H. et al. Biochem J 414, 161 (2008).
6. Setsuie, R. et al. Neurochem Int. 51, 105 (2007).
7. Hussain, S. et al. Cell Cycle 8, 1688 (2009).
8. Nicholson, B. et al. Future Onco l3, 191 (2007).
9. Colland, F. Biochem Soc Trans 38, 137 (2010).
AnaSpec, EGT Group
AnaSpec (anaspec.com), EGT Group is a leading provider of integrated proteomics and genomics solutions™ for worldwide life science research. AnaSpec offers expertise in peptides, detection reagents, antibodies, assay kits, amino acids, oligonucleotides, and qPCRs. AnaSpec carries a broad product line of biochemicals and reagents for basic research, high-throughput screening and drug discovery. AnaSpec also provides premier custom services including peptide synthesis, antibody production, assay development, fluorescent dyes, and oligonucleotide synthesis. AnaSpec is certified to ISO 9001:2008.
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