REEAD TOP1 Activity Kit
225.00 €+ excl. VAT
The VPCIR ™ REEAD TOP1 Activity Kit is designed for assaying eukaryotic TOP1 activity and drug sensitivity. They can be used with purified TOP1 enzyme or cell/tissues extracts.
Research in Cancer, Research in Chronic diseases, Research in Infectious diseases
21 test – 1 WellMaker, 42 test – 2 WellMakers
RED fluorescent dyes, GREEN fluorescent dyes
Not IATA restricted
Kit components must be stored under different conditions – see below.
The VPCIRTM REEAD TOP1 Activity Kit, is designed to allow for an easy, sensitive, specific, and quantitative detection of type 1 topoisomerase activity. REEAD stands for Rolling circle Enhanced Enzyme Activity Detection. The assay is based on the topoisomerase 1 ability to cleave and ligate DNA, converting a DNA substrate into a closed circle (1 and 2). The generated circles are hybridized to a functionalized glass slide and subsequently amplified by Rolling Circle Amplification (3) mediated by the phi29 polymerase. The amplification give rise to fluorescent rolling circle products, allowing for the visualization in a fluorescent scanner or fluorescent microscope (4). This allows for the quantitative measurements of 20+ samples faster than the state-of-the-art gel-based relaxation assay, giving reproducible one-day results.
APPLICATION Eukaryotic topoisomerase 1 are among the DNA binding enzymes with high biological relevance, because they are targets of small-molecule drugs with potential activity against human cancers or pathogens causing diseases in humans. This kit provides a fast, reliable, and quantitative drug-screening tool. It allows to screen the efficiency of libraries of natural or synthetic small-molecule compounds as potential inhibitors of eukaryotic topoisomerase 1. Moreover, since the conversion of the DNA substrate to a closed circle relies specifically on the activity of topoisomerase 1, the assay can be used to measure enzyme activity in small crude biological samples such as cell or tissue extracts in the context of biological or cancer research. KIT CONTENT – A slide mailer containing the functionalized slide(s). – A box containing:
- DNA substrate
- Enzymes (hTOP1 for positive control, phi29)
- Enzymes reactions buffers
- dNTPs, primer and other reagents
- Bottles with buffers
The kit components require different storage conditions:
- The slides are shipped in a slide mailer inside a sealed foil-bag, desiccated. The slide(s) must be stored in the provided bag at 15°C-30°C, until use. Upon opening of the bag, the slide(s) need to be used immediately.
- The enzymes and reagents must be stored at -20°C upon arrival.
- Buffer 1 and Buffer 2 must be stored at 50°C
- Buffer 3 and Buffer 4 need to be stored at 15°C-30°C.
Safety Data Sheet for REEAD TOP1 Activity kits
- Stougaard, M.; Lohmann, J.S.; Mancino, A.; Andersen, F.F.; Koch, J.; Knudsen, B.R. Single-Molecule Detection of Human Topoisomerase I cleavge-Ligation Activity. 2009, 3, 223–233.
- Andersen, F.F.; Stougaard, M.; Jørgensen, H.L.; Bendsen, S.; Juul, S.; Hald, K.; Andersen, A.H.; Koch, J.; Knudsen, B.R. Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level. ACS Nano 2009, 3, 4043–54, doi:10.1021/nn9012912.
- Tesauro, C.; Keller, J.G.; Gromova, I.; Gromov, P.; Frøhlich, R.; Erlandsen, J.U.; Andersen, A.H.; Stougaard, M.; Knudsen, B.R. Different Camptothecin Sensitivities in Subpopulations of Colon Cancer Cells Correlate with Expression of Different Phospho-Isoforms of Topoisomerase I with Different Activities. Cancers (Basel). 2020, 1240, doi:10.3390/cancers12051240.
- Tesauro, C.; Simonsen, A.K.; Andersen, M.B.; Petersen, K.W.; Kristoffersen, E.L.; Algreen, L.; Hansen, N.Y.; Andersen, A.B.; Jakobsen, A.K.; Stougaard, M.; et al. Topoisomerase I activity and sensitivity to camptothecin in breast cancer-derived cells : a comparative study. BMC Cancer 2019, 1–15.
- Alonso, C.; Fuertes, M.; Martín-Encinas, E.; Selas, A.; Rubiales, G.; Tesauro, C.; Knudssen, B.K.; Palacios, F. Novel topoisomerase I inhibitors. Syntheses and biological evaluation of phosphorus substituted quinoline derivates with antiproliferative activity. Eur. J. Med. Chem. 2018, 149, doi:10.1016/j.ejmech.2018.02.058.
- Pommier Editor, Y. DNA Topoisomerases and Cancer; Press, H., Ed.; 2011;
- Keller, J.G.; Tesauro, C.; Coletta, A.; Graversen, A.D.; Ho, Y.-P.; Kristensen, P.; Stougaard, M.; Knudsen, B.R. On-slide detection of enzymatic activities in selected single cells. Nanoscale 2017, 9, doi:10.1039/c7nr05125e.
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