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Stock availability
In Stock
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Kit s description
Fluorometric detection of purified 20S proteasome activity
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Protein target
20S Proteasome
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Scientific context
Proteasomes are non-lysosomal proteolytic complexes localised primarily in the cytoplasm and in the nucleus of eukaryotic cells. They are responsible for the ubiquitin-mediated degradation of short half-life proteins and peptides that are involved in essential cellular processes including cell-cycle regulation, apoptosis and transcriptional regulation, innate immunity and antigen processing, and in the removal of redundant or damaged proteins. As such protein degradation by the ubiquitin-proteasome pathway has a major regulatory function for proliferation activity and survival of both normal and malignant cells, and its dysfunction has been implicated in a wide range of other disease processes including neurodegenerative, cardiovascular and metabolic disorders. The 26S proteasome structure is composed of a 20S proteasome catalytic core complex and one or two 19S (PA700) regulatory subcomplexes. The 20S core comprises two copies of 14 subunits (7 alpha subunits and 7 beta subunits) arranged in a α7β7β7α7 cylindrical array. Proteolytic activities are determined by the β1 (caspase-like), β2 (trypsin-like) and β5 (chymotrypsin-like) subunits, access to which is guarded by the α-subunits. The 19S regulatory unit consists of six ATPase and at least ten non-ATPase subunits that are required for ubiquitinated protein binding, deubiquitination, substrate unfolding and translocation to the 20S catalytic core. Varying catalytic subunit composition (β1, β1i; β2, β2i; β5, β5i) results in a variety of possible subtypes from full constitutive proteasome (β1, β2, β5) through mixed populations to full inducible / immunoproteasome (β1i, β2i, β5i). Alternative regulatory complexes such as the PA200 and 11S proteasome activators confer different substrate specificities and activity compared to the 19S regulator.
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Primary research area
Cancer, Apoptosis, Cell Signaling, Post-translational Modifications, Neuroscience, Neurodegeneration, Cardiovascular System
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Category
Assay Activity Kits
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Other name
Chymotrypsin-like (β5) 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 proteasome inhibitors and activators, and to measure and quantify 20S proteasome activity in continuous kinetic and end-point assays.
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Brief protocol
The 20S Proteasome Activity kit facilitates the rapid, robust measurement of proteasome activity in vitro. The kit utilises high purity, fluorogenic substrate Suc-LLVY-AMC together with suitable calibration standards and controls for the accurate and sensitive assessment of proteasome chymotrypsin-like (β5) activity. Continuous kinetic or end-point assays can be performed in micro-cuvettes or in 96-well plate format for multi-sample analysis. Contains sufficient materials for one full 96-well plate assay or up to 100x 50µL micro-cuvette based assays to be run.
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Kit contents
10x StressXpress® proteasome assay buffer, 20S proteasome, MG132 (control inhibitor), StressXpress® 96 well assay plate, StressXpress® AMC standard, Suc-LLVY-AMC (chymotrypsin-like substrate)
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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 using the plate format, or 100x 50µL micro-cuvette based assays
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Assay duration
15-30 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
4°C
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Shipping recommendations
Blue Ice
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Bibliography
1. Saeki, Y. & Tanaka, K. Methods in molecular biology (Clifton, N.J.) 832, 315–37 (2012). 2. Frankland-Searby, S. & Bhaumik, S. R. Biochimica et biophysica acta 1825, 64–76 (2012). 3. Basler, M., Kirk, C. J. & Groettrup, M. Current opinion in immunology 1–7 (2012). 4. Löw, P. General and comparative endocrinology 172, 39–43 (2011). 5. Dennissen, F. J. a, Kholod, N. & Van Leeuwen, F. W. Progress in neurobiology 96, 190–207 (2012). 6. Geng, F., Wenzel, S. & Tansey, W. P. Annual review of biochemistry 81, 177–201 (2012).
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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
AMC Standard Curve for 20S Proteasome Activity Kit Gold. Representative plot of fluorescence measurements (RFU) of serial dilutions of AMC Standard at recommended concentrations. | Continuous kinetic enzyme activity assay showing the time course of control 20S proteasome activity with A) Suc-LLVY-AMC, B) Z-LLE-AMC and C) Bz-VGR-AMC using the StressXpress® 20S Proteasome Activity Kit GOLD. In each case 20S proteasome (~2.5nM) was incubated with 100µM substrate in proteasome assay buffer for 30 minutes at room temperature alongside a substrate only (blank) control reaction. Fluorescence measurements (RFU) were taken at 30 second intervals and plotted vs. time. Graph of the AMC Typical Standard Curve for the 20S Proteasome Activity Kit GOLD StressXpress - SKT-134 | Enzyme Activity Graph of the Continuous Kinetic Assay for the 20S Proteasome Activity Kit GOLD StressXpress - SKT-134
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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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