REEAD Activity Kit + 1.5 µg Human TOP1 Enzyme
575.00 € excl. VAT
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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 (figure; 1 and 2). The generated circles are hybridized to a functionalized glass slide and subsequently amplified by Rolling Circle Amplification (RCA, figure, 3) mediated by the phi29 polymerase. The amplification give rise to fluorescent rolling circle products, allowing for the visuialization in a fluorescent scanner or fluorescent microscope (figure, 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.
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.
– 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
• Info card with QR code for manual
Manual is coming soon.
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.
- Blocking buffer and Wash buffer 1 must be stored at 50°C
- Wash buffer 2 and Wash buffer 3 need to be stored at 15°C-30°C.
Safety Data Sheet for REEAD TOP1 Activity kits
1. Blanco, L.; Bernad, A.; Lázaro, J.M.; Martín, G.; Garmendia, C.; Salas, M. Highly Efficient DNA Synthesis by the Phage ϕ 29 DNA Polymerase. J. Biol. Chem. 1989, 264, 8935–8940, doi:10.1016/s0021-9258(18)81883-x.
2. Yokouchi, H.; Fukuoka, Y.; Mukoyama, D.; Calugay, R.; Takeyama, H.; Matsunaga, T. Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using j 29 polymerase. 2006, 8, 1155–1163, doi:10.1111/j.1462-2920.2006.01005.x.
3. Larsson, C.; Koch, J.; Nygren, A.; Janssen, G.; Raap, A.K.; Landegren, U. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. 2004, 1, 1–6, doi:10.1038/NMETH723.
4. Salas, M.; Blanco, L.; Lázaro, J.M.; De Vega, M. The bacteriophage φ29 DNA polymerase. IUBMB Life 2008, 60, 82–85, doi:10.1002/iub.19.
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