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Quinolones such as ciprofloxacin are widely used antibiotics that target bacterial DNA topoisomerases. The S83W and D87G mutations in the gyrA subunit of E. coli gyrase cause quinolone resistance, so new molecules that inhibit the mutant enzymes are needed.
This kit measures the DNA supercoiling activity of the quinolone-resistant S83W gyrase mutant, so it can be used to test the inhibitory activity of new quinolone compounds against it.
Kit Components
| Component | S83W100KE |
|---|---|
| 10 x Buffer T2 | 600 µl |
| 10 x Relaxed DNA | 405 µl |
| 1500 x H19 dye | 20 µl |
| 10 x ATP | 450 µl |
| 10 x H19 dilution buffer | 3000 µl |
| 100 x E. coli gyrase mutant S83W | 50 µl |
Final reaction conditions: 20 mM Tris-HCl pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 25 µg/ml relaxed plasmid DNA, 1 mM ATP and 50 nM gyrase. Magnesium is essential; EDTA should be avoided.
Assay Principle
Relaxed and supercoiled DNA give different fluorescence with the H19 dye: in the presence of magnesium, relaxed DNA suppresses the dye signal much more than supercoiled DNA. As gyrase converts relaxed plasmid into its supercoiled form, fluorescence (Ex 485 nm / Em 535 nm) rises; inhibitors keep the signal low.
Specifications
| Assay Target | E. coli gyrase S83W supercoiling |
|---|---|
| Target Type | DNA Topoisomerase II (Gyrase) |
| Organism | E. coli |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening Assay |
| Detection Method | Fluorescence |
| Detection Wavelength | Ex 485 nm / Em 535 nm |
| Plate Format | 96-well plate |
| Instrument Required | Fluorescence microplate reader |
| Assay Time | About 65 min (60 min reaction + 5 min dye incubation) |
| Mutation | S83W in the gyrA subunit (quinolone-resistant) |
| 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.
- Quinolone-resistant E. coli Gyrase Mutant Assays (PDF, see Documents): Covers catalog # S83W100KE.
Safety & Handling
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