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Type II topoisomerases, which include mammalian topoisomerase II, bacterial gyrase and topoisomerase IV (parC-parE), alter DNA topology by cutting both strands and joining them again. If a compound such as a fluoroquinolone stops the rejoining, the cut (linear) DNA stays covalently linked to the enzyme as a cleavage complex, which harms DNA in cells (a topoisomerase poison).
Checking for cleavage complex formation helps show how a new topoisomerase inhibitor acts. Novobiocin and ciprofloxacin both inhibit DNA gyrase, for example, yet only ciprofloxacin leads to cleavage complexes.
Kit Components
| Component | TDC96K |
|---|---|
| 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 |
Topoisomerase is not included. A plate vacuum filtration device and a lab vacuum line (up to 80 kPa or 600 mmHg) are needed and are not provided.
Assay Principle
After the topoisomerase reaction, the protein is removed with SDS and proteinase K and the sample is mixed with a matrix. Linearized DNA passes through the matrix and filter plate much more easily than closed circular DNA; the filtered DNA is collected in a black plate, stained with a dye and read at 535 nm (excitation 485 nm), so cleavage-complex formers raise the signal.
Specifications
| Assay Target | Topoisomerase DNA cleavage complex |
|---|---|
| Target Type | Topoisomerase |
| Assay Type | 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 |
| Assay Time | About 130 min (60 min reaction + 60 min proteinase K + 5 min matrix mix + about 5 min filtration) |
| Equipment Needed | Lab vacuum line or pump (up to 80 kPa / 600 mmHg), plate vacuum device, fluorescence plate reader |
| 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 # TDC96K.
Safety & Handling
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