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DNA replication requires decatenation, the separation of interlinked DNA circles. In bacteria this is done by topoisomerase IV (the parC-parE complex), which turns concatenated DNA into decatenated DNA. The enzyme is a target for antibacterial inhibitor tests.
Kit Components
| Component | TDD96K |
|---|---|
| 10 x Assay buffer | 600 µl |
| 10 x Concatenated DNA | 500 µl |
| 10 mM ATP | 110 µl |
| 0.4 M EDTA | 600 µl |
| 20 x Fluorescence dye | 260 µl |
| 10 x Rinse buffer | 2 ml |
| V-bottom plate | 1 plate |
| TDD filter plate | 1 plate |
| Black 96-well plate | 1 plate |
The assay buffer is optimized for bacterial topoisomerase IV; the decatenation enzyme is not included. The kit can be used with any bacterial topoisomerase IV (parC-parE) or gyrase. Final reaction conditions: 20 mM Tris-HCl, pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 3 µg/ml concatenated DNA and 0.2 mM ATP. A plate vacuum device and vacuum line (up to 80 kPa or 600 mmHg) are needed and not provided.
Assay Principle
After the decatenation reaction, the sample is filtered through the TDD filter plate. Decatenated DNA passes the filter into a black 96-well plate while concatenated DNA is retained; the passed DNA is stained with a fluorescence dye and read at 535 nm (excitation 485 nm), so inhibitors lower the signal.
Specifications
| Assay Target | Topoisomerase DNA decatenation |
|---|---|
| Target Type | Topoisomerase |
| 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 / 600 mmHg), plate vacuum device, fluorescence plate reader |
| Enzyme Compatibility | Bacterial topoisomerase IV (parC-parE) or gyrase |
| Size | 96 assays |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- 96-Well Topo IV DNA Decatenation Assay (P. aeruginosa Topo IV and general kits) (PDF, see Documents): Covers catalog # TDD96K.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this topoisomerase DNA decatenation assay — including larger pack sizes, bulk reagents, or a protocol adapted to your samples. Click Talk to a Scientist, email support@biohippo.com, or explore our Research Services. Our team will be in touch shortly.
