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| Product Name | 20S Proteasome Activity Kit |
| Description |
Fluorometric detection of purified 20S proteasome activity |
| Species Reactivity | Species Independent |
| Platform | Microplate |
| Sample Types | Purified Proteins |
| Detection Method | Fluorometric Assay |
| Assay Type | Continuous Kinetic Assay |
| Utility | 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. |
| Incubation Time | 15-30 minutes |
| Number of Samples | 40 samples in duplicate using the plate format, or 100x 50µL micro-cuvette based assays |
| Other Resources | Kit Booklet , MSDS |
| Field of Use | Not for use in humans. Not for use in diagnostics or therapeutics. For in vitro research use only. |
Properties
| Storage Temperature | 4ºC | |||||||||||||||||||||
| Shipping Temperature | Blue Ice | |||||||||||||||||||||
| Product Type | Activity Kits | |||||||||||||||||||||
| Assay Overview | 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. | |||||||||||||||||||||
| Kit Overview |
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| Cite This Product | 20S Proteasome Activity Kit (StressMarq Biosciences Inc., Victoria BC CANADA, Catalog # SKT-133) |
Biological Description
| Alternative Names | Chymotrypsin-like (β5) Activity Kit |
| Research Areas | Apoptosis, Cancer, Cardiovascular System, Cell Signaling, Neurodegeneration, Neuroscience, Post-translational Modifications |
| Scientific Background | 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. |
| References |
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). |
Product Images
AMC Standard Curve for 20S Proteasome Activity Kit. 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 Suc-LLVY-AMC substrate using the StressXpress® 20S Proteasome Activity Kit. 20S proteasome (~2.5nM) was incubated with 100µM Suc-LLVY-AMC 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.
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