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Stock availability
In Stock
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Kit s description
Fluorometric detection of DUB activity
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Protein target
DUB
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Scientific context
Conjugation of ubiquitin to proteins (ubiquitination) plays a fundamental role in the regulation of cellular function through biological events involving, amongst others, cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis. The ubiquitin signaling system includes a large family of cysteine proteases known as deubiquitinating enzymes (DUBs) that are responsible for the removal of ubiquitin from modified proteins. This regulatory process allows optimal levels of cellular ubiquitin to be maintained by recycling ubiquitin attached to inappropriate targets, removing and disassembling polyubiquitin chains, and processing proteins prior to their degradation by the proteasome. DUBs in general exhibit a wide range of substrate, ubiquitination type (mono- or poly-ubiquitination) and polyubiquitin chain linkage specificities and can be partnered with various interacting proteins to facilitate increased diversity in specificity and DUB activation. DUBs have been implicated in a number of human diseases including various forms of cancer and neurodegeneration. As such they are attractive targets for potential therapeutic intervention via the development of suitable inhibitors and modulators.
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Primary research area
Cell Signaling, Post-Translational Modifications, Ubiquitination, Epigenetics, Cancer, Apoptosis, Autophagy, Neuroscience, Neurodegeneration, Cardiovascular System
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Category
Assay Activity Kits
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Other name
Deubiquitinating enzyme Activity Kit
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Brand name
StressXpress®
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Detection system
Fluorometric Assay
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Assay format
Continuous Kinetic Enzyme Activity Assay
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Main intended usage
Activity kit used to screen potential inhibitors and activators against DUB enzymes, and to assess and evaluate performance of DUB activity in purified protein samples. It can also be used to optimize assays for specific DUBs to facilitate their use in HTS, and demonsrate that novel DUB enzymes have ubiquitin-AMC processing activity.
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Brief protocol
The DUB Activity kit facilitates the rapid, robust measurement of deubiquitinating enzyme activity in vitro. The kit utilises high purity, fluorogenic substrate ubiquitin-AMC together with suitable calibration standards and controls for the accurate and sensitive assessment of DUB activity. Continuous kinetic or end-point assays can be performed in 96-well plate format for multi-sample analysis. Contains sufficient materials for one full 96-well plate assay set-up to be run.
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Kit contents
10x StressXpress® DUB assay buffer, Control DUB enzyme (USP2 catalytic domain), Ubiquitin-AMC, StressXpress® 96 well assay plate, StressXpress® AMC standard
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Assay precision
Contact us
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Assay cross reactivity
Species Independent
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Samples to be used with this kit
Purified Proteins
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Maximum samples to be used with this kit
40 samples in duplicate
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Assay duration
30-90 minutes
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Kit s sensitivity
See product datasheet or feel free to contact us
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Assay detection limit
See product datasheet or feel free to contact us
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Storage recommendations
-80
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Shipping recommendations
Dry Ice
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Bibliography
1. Komander, D. & Rape, M. Annual Review of Biochemistry 81, 203–229 (2012). 2. Ciechanover, A. Biochimica et biophysica acta 1824, 3–13 (2012). 3. Clague, M. J., Coulson, J. M. & Urbé, S. Journal of cell science 125, 277–86 (2012). 4. Komander, D., Clague, M. J. & Urbé, S. Nature reviews. Molecular cell biology 10, 550–63 (2009). 5. Fraile, J. M., Quesada, V., Rodríguez, D., Freije, J. M. P. & López-Otín, C. Oncogene 31, 2373–88 (2012). 6. Kessler, B. M. Current opinion in chemical biology 17, 59-65 (2013). 7. Bilguvar, K. et al. Proceedings of the National Academy of Sciences of the United States of America 110, 3489–94 (2013).
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Release date
1-Jun-2014
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PubMed number
Refer to PubMed
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Tested applications
No data available / currently testing
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Tested reactivity
No data available / currently testing
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Resources available upon request
Kit Booklet, MSDS
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Product image link
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Product image legend
Continuous kinetic enzyme activity assay showing the time course of control DUB enzyme (USP2 CD) activity with Ub-AMC substrate using StressXpress® DUB Activity Assay kit – Control DUB (10nM) was incubated with 500nM Ub-AMC in DUB assay buffer for 15 minutes at room temperature alongside a substrate only (blank) control reaction. Fluorescence measurements (RFU) were taken at 30 second intervals and plotted vs. time | AMC Standard Curve for StressXpress® DUB Activity Assay Kit. Representative plot of fluorescence measurements (RFU) of serial dilutions of AMC Standard at recommended concentrations. Enzyme Activity Graph of the Continuous Kinetic Assay for the DUB Activity Kit StressXpress - SKT-136 | Graph of the AMC Typical Standard Curve for the DUB Activity Kit StressXpress - SKT-136
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Warnings
Non-hazardous
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Country of production
Canada
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Total weight kg
2
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Net weight g
300
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