| Field | Specification |
|---|---|
| Organism | |
| Applications | |
| Assay type | |
| Assay target | |
| Assay area | |
| Target type | |
| Detection method | |
| Detection wavelength | |
| Instrument required | |
| Plate format | |
| Storage | |
| Shipping | |
| Catalog no. (Mfr.) | |
| Main SKU |
Type II topoisomerases include bacterial gyrase, mammalian topoisomerase II and bacterial topoisomerase IV (parC-parE). They alter DNA topology by cutting both DNA strands and joining them again.
Some inhibitors, such as fluoroquinolones, block the rejoining step. The cut (linear) DNA then stays bound to the enzyme as a covalent cleavage complex, which builds up and damages DNA in cells (topoisomerase poison). Ciprofloxacin and novobiocin both inhibit gyrase, yet only ciprofloxacin forms this complex, so testing for it helps show how new inhibitors act.
Kit Components
| Component | T4C96KE |
|---|---|
| 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 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 | E. coli Topo IV DNA cleavage |
|---|---|
| Target Type | DNA Topoisomerase IV |
| 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 # T4C96KE.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this E. coli Topo IV DNA cleavage assay — including larger pack sizes, bulk reagents, bulk enzyme, 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.
