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Human Topoisomerase II DNA Decatenation Assay Kit (enzyme not included)

BHE16300015

This product includes the assay buffer, concatenated DNA, spin columns and loading solution for 20 assays of DNA decatenation reactions. The assay buffer is optimized for human topoisomerase II alpha.

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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 Protocol for spin column-based DNA decatenation assay
1. Reaction and sample preparation:
(1) 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 (10 U/ µl) solution 10 fold using the dilution buffer, Step 3, incubate the reaction mixture at 37°C for 60 min.
 
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.
 
(2) Add 5 µl 0.5 M EDTA to stop the reaction.
 
2. Column preparation:
(1) Spin the column at 13000 rpm using a bench top Eppendorf centrifuge for 30 seconds to set down the resin.
(2) Remove the column cap and bottom tip. Cut off the cap of a 1.5-Eppendorf tube. Place the column into the tube. Spin the column at 13000 rpm for 2 min. Transfer the column into a fresh Eppendorf tube.
 
3. Assay
(1) Load the 50 µl of the reaction mixture onto the column. Spin the column at 13000 rpm for 2 min. Collect the eluted solution from the spin column. Transfer the solution into a standard black 96-well plate well.
(2) Dilute the 20 x fluorescence dye with water to make the 1 x fluorescence dye. Mix 150 µl of the 1x fluorescence dye with the column eluent.
(3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
 
96-well DNA decatenation assays
DNA decatenation assays in a 96-well plate format are available for DNA decatenation enzymes including human topoisomerase II and bacterial topoisomerase IV.
Combination SKU Supplier No. Specifications Size Your price Quantity
BHE16300015-1 HDC020K enzyme not included 20 assays $250.74
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BHE16300015-2 HDC020KE enzyme included 20 assays $327.89
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BHE16300015-3 HDC100KE enzyme included 100 assays $1173.18
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