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Thymidylate kinase phosphorylates dTMP with ATP, giving dTDP and ADP. This step is part of the dTTP synthesis pathway that feeds DNA synthesis. Tumors show higher activity of this enzyme than the matching normal human tissue.
Kit Components
| Component | HTMK500KE |
|---|---|
| 10 x Reaction buffer | 1700 µl |
| 2500 x human thymidylate kinase (125 µM) | 8 µl |
| 100 x ATP (5 mM) | 170 µl |
| 100 x dTMP (40 mM) | 170 µl |
| 100 x MUK Reagent A | 170 µl |
| 100 x MUK Reagent B | 170 µl |
| 10 x Fluorescence dye | 1700 µl |
1 x reaction buffer: 50 mM Tris-HCl, pH 8.0, 3 mM MgCl2, 0.2 mM EDTA, 50 mM NaCl, 0.003% Brij-35.
Assay Principle
The assay measures the ADP generated by the kinase reaction. Reagents A and B are added after the reaction starts, and a fluorescence dye is then read at 535 nm with excitation at 485 nm, in black or white 96-well or 384-well plates or micro-cuvettes.
Specifications
| Assay Target | Human thymidylate kinase activity |
|---|---|
| Target Type | Kinase |
| Organism | Human |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening Assay |
| Detection Method | Fluorescence |
| Detection Wavelength | Ex 485 nm / Em 535 nm |
| Plate Format | 384/96-well plate or micro-cuvette |
| Instrument Required | Plate reader or fluorometer |
| Assay Time | About 62 min (2 min pre-incubation + 60 min reaction) |
| Size | 500 assays (384-well) or 300 assays (96-well) |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- Human Thymidylate Kinase Assay (PDF, see Documents): Covers catalog # HTMK500KE.
Safety & Handling
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