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Human thymidylate kinase (TMK) - for 500 assays

BHE16300233

This product includes 20 ul of 50 uM human thymidylate kinase.

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+1 866.986.9598
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[email protected]
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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).  
Product Manual Datasheet
Protocols Description 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.
SKU Supplier No. Size Your price Quantity
BHE16300233 HTMK-500 500 assays $1101.97
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