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Home / Assay Kits

96-Well Human Topo II DNA Decatenation Assay Kits

SKU : BHE16300013

This product includes the assay buffer, concatenated DNA, human topo II alpha, ATP, fluorescence dye, rinse buffer and a TDD filter plate for 96 assays of human topo II DNA decatenation reactions.

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Mfr.No.	HDD96KE, TDD96K
Description	DNA decatenation is an essential process during DNA replication in the cells. The DNA decatenation reaction was carried out by topoisomerase II in human. The DNA decatenation reaction converts the concatenated DNA into decatenated DNA. The 96-Well Topoisomerase DNA Decatenation Assay is in a 96-well assay plate format that can be used for high-throughput tests of topoisomerase inhibitors. The assay is based on the principle that the decatenated DNA is separated from the concatenated DNA by a filtration process. The decatenated DNA passed through the filter (TDD filter plate), received in a black 96 well plate and quantified by fluorescence at 535 nm (excitation at 485 nm). Each 96-Well Human Topo II DNA Decatenation Assay Kit Plus (Catalog No. HDD96KE) includes 600 µl of 10 x assay buffer, 500 µl of 10 x concatenated DNA, 520 µl of 2 mM ATP, 26 µl of 200 x human Topo II, 500 µl of enzyme dilution buffer, 1 ml of Stop solution (0.4 M EDTA), 530 µl of 20 x fluorescence dye, 2 ml of 10 x rinse buffer, one V-bottom plate, a TDD filter plate and one black 96-well plates for 96 assays of DNA decatenation reactions.
Drug target assays	Anticancer drug targets

Reference	1. Abderrazzak Merzouki et al, Adva-27a, a Novel Podophyllotoxin Derivative Found to Be Effective against Multidrug Resistant Human Cancer Cells, Anticancer Research 32: 4423-4432 (2012). 2. Narayanan S. et al, A cell cycle-controlled redox switch regulates the topoisomerase IV activity. Genes Dev. 29(11):1175-87 (2015).

Product Manual	Datasheet
Protocols Description	Assay Protocol 1. Reagent preparation and filtration unit Dilute the 20 x fluorescence dye 20-fold to make 1x fluorescence dye. Dilute the 10 x Rinse buffer 10-fold with water to make 1 x Rinse buffer. Assembly the filtration unit by connecting the filtration device to a vacuum line, placing the black 96-well plate in the chamber of the filtration device as a receiver of the filtration and the TDD filter plate on the top of the device. 2. Reaction and sample preparation: The reactions are carried out in a V-bottom plate. The total volume of each reaction mixture is 50 µl. Step 1: mix 32.5 µl of H2O, 5 µl of 10 x assay buffer, 5 µl of 10 x concatenated DNA and 5 µl of 2 mM ATP. Step 2, add 2.5 µl of 20 x topoisomerase (1 U/µl) which is freshly diluted from the 200 x stock solution 10 fold using the dilution buffer, Step 3, incubate the reaction mixture at 37°C for 60 min. At the end of the reaction, add 5 µl of Stop Solution to stop the reaction. Note: The final concentrations are 10 mM Tris-HCl, pH 8, 50 mM NaCl, 0.1 mM EDTA, 50 mM KCl, 5 mM MgCl2, 15µg/ml BSA, 3 µg/ml concatenated DNA, 0.2 mM ATP and 50 U/ml human topoisomerase II alpha. A lower temperature will have a slower reaction. For IC50 experiments, enzyme dose response should be tested to avoid using too much enzyme. The enzyme must be diluted in the enzyme dilution buffer freshly. Do not store the diluted enzyme. 3. Assay Load 50 µl of the sample onto the filter plate. Apply the vacuum (80 kpa or 600 mmHg) until the solution goes though the filter. Add 150 µl of the Rinse Buffer and let the buffer completely go through the filter. Stop the vacuum and take out the receiver plate. Add 50 µl of the 1 x dye into each well. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.

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Combination SKU	Supplier No.	Specifications	Your price	Quantity
BHE16300013-1	HDD96KE	enzyme included	$862.85		Add to Cart
BHE16300013-2	TDD96K	enzyme not included	$658.26		Add to Cart

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