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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, trypsin-like (β2) and caspase-like (β1) 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 GOLD facilitates the rapid, robust measurement of purified proteasome activity. The kit utilises high purity, fluorogenic substrates Suc-LLVY-AMC, Bz-VGR-AMC, and Z-LLE-AMC together with suitable calibration standards and controls for the accurate and sensitive assessment of proteasome chymotrypsin-like (β5), trypsin-like (β2) and caspase-like (β1) activities. 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 two full 96-well plate assays or up to 200x 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), Bz-VGR-AMC (trypsin-like substrate), Z-LLE-AMC (caspase-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
80 samples in duplicate using the plate format, or 200x 50µL micro-cuvette based assays
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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
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
Experimental timeline for StressXpress® Autophagy Flux Detection Kit sample preparation. | Schematic showing basal autophagy in cells and the effect of bafilomycin A1 (BafA1) treatment. Difference between LC3-II or p62 marker levels with and without BafA1 treatment represents the autophagic flux/turnover of the system and can be used to assess changes in autophagy upon experimental investigation. | Western blot showing detection of LC3-II, p62 and actin in untreated control cells (UT), starved cells (ST) and cells treated with a potential autophagy modulator (MT) and in the absence and presence of bafilomycin A1 (BafA1) using the StressXpress® Autophagy Flux Detection Kit. | Western blot diagram showing comparison of LC3-II and p62 marker levels upon treatment with autophagy modulators resulting in activation (1), late stage inhibition (2) or early stage inhibition (3) of autophagy. | Measurement of change in autophagic flux (ΔAF) in cells following starvation or modulator treatment. | Data showing LC3-II and p62 levels (normalised to actin) for untreated control (UT), starved (ST) and modulator treated (MT) cells derived from Western blot band intensities (Figure 3). Diagram of the Experimental timeline for the Autophagy Flux Detection Kit StressXpress - SKT-135 | Diagram of the Basal Autophagy and the effect of bafilomycin A1 for the Autophagy Flux Detection Kit StressXpress - SKT-135 | Western Blot of the Example Results for the Autophagy Flux Detection Kit StressXpress - SKT-135 | Western Blot of the Example results of LC3-II and p62 marker levels after modulation for the Autophagy Flux Detection Kit StressXpress - SKT-135 | Sample Results Graph of the Change in Autophagic Flux for the Autophagy Flux Detection Kit StressXpress - SKT-135 | Sample Results Graph of the LC3-II and p62 levels for the Autophagy Flux Detection Kit StressXpress - SKT-135
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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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