Topoisomerase II Drug Screening Kit (kDNA based) Product Description
This Kit will unambiguosly identify compounds that target topoisomerase II action (topo II). The kit detects two kinds of Top2 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. Plasmid based kits, such as TG1009, are 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, Top2 enters into a breakage/resealing cycle that favors the resealed product (Fig 1). The cleavage intermediate has an extremely short lifetime and cannot be identified. A known Top2 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 (kDNA) included in this assay is ideal because it 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 Top2. In this case, there is a clear loss of decatenated kDNA products. The above video has more information.
Topo II Drug Screening Kit Contents (for 100 assay kit size):
-100 Units Human Top2a (available with catalog no. TG1019-1A)
-Kinetoplast DNA (kDNA) 20 ug
-Decatenated and Linearized kDNA Markers
-10X Topoisomerase II assay buffer components*
*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.
-Sodium dodecyl sulfate (10%)
-10X Gel loading buffer (bromophenol blue, glycerol)
-Detailed instruction manual
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.