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Type II DNA topoisomerases use ATP hydrolysis to drive DNA supercoiling (gyrase) and decatenation (topoisomerase IV). Blocking this ATPase activity, as novobiocin does, also blocks the enzyme's biological role, which makes the P. aeruginosa Topo IV ATPase reaction a useful readout for screening topoisomerase inhibitors.
Kit Components
| Component | T4A-100KP |
|---|---|
| 10 x Buffer | 400 µl |
| 100 x DNA | 35 µl |
| 100 x P. aeruginosa Topo IV | 33 µl |
| 100 x ATP | 35 µl |
| Phosphate detection dye | 5 ml |
The 100 x DNA stock is 1 mg/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.
Assay Principle
The kit detects the inorganic phosphate released when topoisomerase IV hydrolyzes ATP in the presence of DNA. A phosphate detection dye is added after the reaction and absorbance is read at 650 nm; inhibitors lower the signal.
Specifications
| Assay Target | P. aeruginosa Topo IV ATPase |
|---|---|
| Target Type | Topoisomerase |
| Organism | P. aeruginosa |
| 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 # T4A-100KP.
Safety & Handling
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