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Type II DNA topoisomerases, such as gyrase (topoisomerase II) and topoisomerase IV, draw energy from ATP hydrolysis to supercoil and decatenate DNA. Compounds like novobiocin that block this ATPase activity also stop the enzyme's biological role. For this reason, an ATPase assay suits high-throughput screening for topoisomerase inhibitors.
Kit Components
| Component | T2A-100KS |
|---|---|
| 10 x Buffer | 400 µl |
| 100 x DNA | 35 µl |
| 100 x S. aureus gyrase | 33 µl |
| 100 x ATP | 35 µl |
| Phosphate detection dye | 5 ml |
Final reaction conditions: 20 mM Tris-HCl pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 10 µg/ml DNA, 0.2 mM ATP and 50 nM topoisomerase. A reaction without enzyme can serve as negative control.
Assay Principle
The kit detects the inorganic phosphate released when gyrase hydrolyzes ATP in the presence of DNA. A phosphate detection dye is added and absorbance at 650 nm is read; inhibitors of the ATPase lower the signal.
Specifications
| Assay Target | S. aureus gyrase ATPase activity |
|---|---|
| Target Type | DNA Topoisomerase II (Gyrase) |
| Organism | S. aureus |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening Assay |
| Detection Method | Colorimetric |
| Detection Wavelength | Absorbance 650 nm |
| Plate Format | 384-well plate |
| Instrument Required | Absorbance microplate reader |
| Assay Time | About 60 min |
| Size | 100 assays |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- DNA Topoisomerase ATPase Assays (PDF, see Documents): Covers catalog # T2A-100KS.
Safety & Handling
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