The Topoisomerase II Drug Screening Kit (kDNA based) will unambiguously identify compounds that target topoisomerase II action (Human Topoisomerase IIa Enzyme). The kit detects two kinds of Topo II effectors: those that inhibit the activity of the enzyme (Catalytic Inhibitory Compounds or CICs) and those that stimulate formation of the cleavage complexes (Interfacial Poisons or IFPs). CICs may affect enzyme at one of many levels, such as blocking access to DNA substrate or altering ATPase action and enzyme turnover. IFPs are another type of inhibitor that blocks the resealing step of the reaction on DNA such that DS DNA breaks tend to accumulate. Our plasmid based Human Topoisomerase II Drug Screening Kit is ideal for detecting IFP (poisons like VP16). This kDNA based Drug Kit is optimized for detecting topo II catalytic inhibitors (CIC) type of drugs. Under normal circumstances, Topo II enters into a breakage/resealing cycle that favors the resealed product. The cleavage intermediate has an extremely short lifetime and cannot be identified. A known Topo II poison (etoposide or VP16) is included as a control IFP since a positive drug is required to ensure that the assay is working properly and is capable of resolving unknown IFP mechanistic drugs. The DNA substrate (kinetoplast DNA) included in this assay is a very attractive substrate for topo II catalysis. This kDNA assay system is based upon evaluating either the formation of DNA cleavage products (linear kDNA) for IFPs. For CIC detection, the assay simply measures inhibition of the decatenase activity of topo II. In this case, there is a clear loss of decatenated kDNA products. The following video has more information.
Topoisomerase II Drug Screening Kit (kDNA based)
Topo II Drug Kit 100 Assay Kit Size
-100 Units Human Top2a (included with catalog no. TG1019-1A only)
-kDNA (20 ug total) substrate at the concentration specified on the tube provided
-Decatenated kDNA marker (25 ul) in gel loading buffer
-Linearized kDNA marker (25 ul) in gel loading buffer
-10X Topoisomerase II assay buffer components*
-VP-16 (etoposide) control drug in lyophilized state
-10% sodium dodecyl sulfate (SDS) (300 ul)
-10x loading buffer (300 ul)
-Proteinase K (500 ul) at 0.5 mg/ml
–Detailed instruction manual
*To improve stability, the 10x Topo II Assay buffer is provided as two components: Buffer A (no ATP) and Buffer B (ATP). The Complete 10x Assay buffer is made fresh each time by combining Buffer A and B.
This kit is shipped at ambient temperature or on dry ice if purchased with enzyme. Store enzyme at -70° C. The DNAs should be stored at 4° C. The assay buffer components should be stored at -20° C upon receipt. The VP-16 can be stored at ambient temperature.
Topoisomerase-II-Drug-Screening-Kit-kDNA Based Kit Protocol
Are you interested in learning about how we can help your lab with your compound screening research? TopoGEN offers powerful and tractable services to identify Topoisomerase-targeting drugs In Vitro. Learn more about these services here.
Reviews and Citations
- Sudan, S., & Rupasinghe, H. V. (2014). Flavonoid-Enriched Apple Fraction AF4 Induces Cell Cycle Arrest, DNA Topoisomerase II Inhibition, and Apoptosis in Human Liver Cancer HepG2 Cells. Nutrition and cancer, ahead-of-print (2014): 1-10. doi: 10.1080/01635581.2014.951733
- Cheng MH, Yang YC, Wong YH, Chen TR, Lee CY, Yang CC, Chen SH, Yang IN, Yang YS, Huang HS, Yang CY, Huang MS, Chiu HF: B1, a novel topoisomerase II inhibitor, induces apoptosis and cell cycle G1 arrest in lung adenocarcinoma A549 cells. Anti-Cancer Drugs 2012, 23(2): 191-199. doi: 10.1097/CAD.0b013e32834cd277
- Shi W, Marcus SL, Lowary TL: Cytotoxicity and topoisomerase I/II inhibition of glycosylated 2-phenyl-indoles, 2-phenyl-benzo[b]thiophenes and 2-phenyl-benzy[b]furans. Bioorganic and Medicinal Chemistry 2010. doi: 10.1016/j.bmc.2010.10.054
- Gong Y, Firestone GL, Bjeldanes LF: 3,3′-Diindolylmethane is a novel topoisomerase IIa catalytic inhibitor that induces S-phase retardation and mitotic delay in human hepatoma HepG2 cells. Molecular Pharmacology 2006, 69(4): 1320-1327.