Protein construct: Wild-type Human Thymidylate Kinase purified from a bacterial protein expression system.
MW: 24 kDa
Enzyme concentration: 50 µM
Enzyme assay: The kinase activity of human thymidylate kinase is measured by using the Human Thymidylate Kinase Assay kit (Catalog No. HTMK500K).
Storage temperature: -20 or -80°C. Do not freeze-and-thaw repeatedly.
Enzyme dilution: Use the 1 x assay to dilute the enzyme just before the assay. Do not store diluted enzyme solution.
The Human Thymidylate Kinase – for 500 assays (Catalog No: HTMK-500) includes 20 µl of 1000 x human thymidylate kinase (50 µM). It is for 500 assays.
Enzymes and Substrates
Bacterial enzymes
Reference
Choi J.Y. et al, Structure Guided Development of Novel Thymidine Mimetics targeting Pseudomonas aeruginosa Thymidylate Kinase: from Hit to Lead Generation, J Med Chem. January 26; 55(2): 852–870 (2012).
Assay Protocol using the Human Thymidylate Kinase Assay kit
The following assay protocol is based on 96-well plate assays (plate type: Costar 3915 or alike), the reaction volume is 50 µl and the final assay volume is 100 µl. For 384-well plate assays (plate type: Matrix 4318 or alike). The reaction volume is 30 µl and the final assay volume is 60 µl.
1. Reagent preparation:
For each 10 assay reactions,
(1) Prepare 55 µl of 10 x ATP by dilution of 5.5 ul of 100 x ATP with 49.5 µl of water.
(2) Prepare 550 µl of 1 x fluorescence dye by dilution of 55 µl of 10 x Fluorescence dye with 495 µl of water.
(3) Prepare 385 µl of premix composed of 324 µl of H2O, 55 µl of 10 x Buffer, 5.5 µl of100 x dTMP and 0.5 µl of 1000 x thymidylate kinase.
2. Kinase assay
In each well,
(1) Mix 35 µl of the premix with 5 µl of 10 x ATP. Incubate the reaction at 37°C for 2 min.
(2) Add 5 µl of 10 x MUK Reagent A and 5 µl of 10 x MUK Reagent B.
(3) Incubate the reaction mixture at 37°C for 60 min.
(4) Add 50 µl of the 1 x fluorescence dye into the 50 µl of the reaction mixture.
(5) Measure the fluorescence intensity at 535 nm with excitation at 485 nm.
Note: The 1 x reaction buffer is 50 mM Tris-HCl, pH 8.0, 3 mM MgCl2, 0.2 mM EDTA, 50 mM NaCl, 0.003% Brij-35. Diluted thymidylate kinase is freshly prepared before the assay.
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