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Bacterial gyrase, mammalian topoisomerase II and bacterial topoisomerase IV (parC-parE) are all type II topoisomerases. They alter DNA topology by cutting both strands and joining them again.
Some inhibitors, such as fluoroquinolones, stop the rejoining step. Cut (linear) DNA then stays bound to the enzyme as a cleavage complex, which damages DNA in cells (a topoisomerase poison). Checking for this complex helps show how a new inhibitor acts: ciprofloxacin and novobiocin both inhibit gyrase, yet only ciprofloxacin leads to cleavage complexes.
Kit Components
| Component | T4C96KS |
|---|---|
| 10 x Reaction buffer | 300 µl |
| 10 x DNA | 30 µl |
| SDS solution | 260 µl |
| 100 mM ATP | 30 µl |
| Proteinase K solution | 260 µl |
| TDC matrix | 10 ml |
| Rinse buffer | 6 ml |
| 10 x Dye | 500 µl |
| Black receiver plate | 1 plate |
| V-bottom reaction plate | 1 plate |
| TDC filter plate | 1 plate |
| 100 x S. aureus Topo IV | 26 µl |
A plate filtration device and a vacuum line are needed for the filtration step (not included).
Assay Principle
Linear DNA passes through a solution matrix and a filter much more easily than closed circular DNA. After the reaction, the sample is treated with SDS and proteinase K, mixed with TDC matrix and filtered on the TDC filter plate. DNA collected in the black receiver plate is stained with dye and read by fluorescence (Ex 485 nm / Em 535 nm), so the signal rises with cleavage complex formation.
Specifications
| Assay Target | S. aureus Topo IV DNA cleavage |
|---|---|
| Target Type | DNA Topoisomerase IV |
| Organism | S. aureus |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening Assay |
| Detection Method | Fluorescence |
| Detection Wavelength | Ex 485 nm / Em 535 nm |
| Plate Format | 96-well filter plate |
| Instrument Required | Fluorescence reader + vacuum device |
| Equipment Needed | Lab vacuum line or pump (up to 80 kPa or 600 mmHg), plate vacuum device, plate fluorescence reader (Ex 485 nm / Em 535 nm) |
| Size | 96 assays |
| Storage | Store the TDC filter plate and TDC matrix at 4 °C and the other reagents at -20 °C. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- Topoisomerase DNA Cleavage Assays (PDF, see Documents): Covers catalog # T4C96KS.
Safety & Handling
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