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Type II topoisomerases (bacterial gyrase, bacterial topoisomerase IV (ParC-ParE) and mammalian topoisomerase II) cut and rejoin both DNA strands to change DNA topology. Some inhibitors, such as fluoroquinolones, block the rejoining step; the enzyme then stays covalently linked to cut (linear) DNA in a cleavage complex, which damages DNA in cells (topoisomerase poisons).
Not every gyrase inhibitor works this way: ciprofloxacin and novobiocin both inhibit gyrase, but only ciprofloxacin produces cleavage complexes. Measuring cleavage complex formation therefore helps show how a new topoisomerase inhibitor acts.
Kit Components
| Component | T2C96KE |
|---|---|
| 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 E. coli gyrase | 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 | E. coli gyrase DNA cleavage |
|---|---|
| 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 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 # T2C96KE.
Safety & Handling
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