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Home / Assay Kits / Drug Target Assays

E. coli Thymidylate Kinase Assay Kit Plus

BHE16300135

This product includes 600 ul of 10 xi Buffer, 35 ul of 100 x dTMP, 35 ul of 100 x E. coli Thymidylate kinase, 35 ul of 100 x ATP, 35 ul of 100 x MUK Reagent A, 35 ul of 100 x MUK Reagent B and 300 ul of 10 x fluorescence dye for 100 assays in a 384-well assay format.

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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
1. Reagent preparation:
(1) 1 x reaction buffer: dilute the 10 x Buffer 10-fold with water. The 1 x buffer is composed of 50 mM Tris[1]HCl, pH 8.0, 3 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 0.003% Brij-35.
(2) 10 x dTMP: dilute the 100 x dTMP solutions (5 mM) 10-fold with water.
(3) 10 x ATP: dilute the 100 x ATP solutions (5 mM) 10-fold with water.
(4) 10 x thymidylate kinase: dilute the 100 x thymidylate kinase 10-fold with 1 x buffer.
(5) 10 x MUK Reagent A: dilute the 100 x MUK Reagent A 10-fold with water.
(6) 10 x MUK Reagent B: dilute the 100 x MUK Reagent B 10-fold with 1 x buffer.
(7) 1 x fluorescence dye: dilute the 10 x Fluorescence dye 10-fold with water.
 
2. Kinase assay
The kinase reaction volume is 30 µl.
(1) In a standard black 384-well plate (Matrix 4318), mix 12 µl of water, 3 µl of 10 x Buffer, 3 µl of 10 x dTMP, 3 µl of 10 x ATP, 3 µl of 10 x kinase. Incubate the reaction for 2 min.
(2) Add 3 µl of 10 x MUK Reagent A, 3 µl of 10 x MUK Reagent B.
(3) Incubate the reaction mixture at 37°C for 60 min.
(4) Add 30 µl of the 1 x fluorescence dye into the 30 µl of the reaction mixture.
(5) Measure the fluorescence intensity at 535 nm with excitation at 485 nm.
Combination SKU Supplier No. Specifications Size Your price Quantity
BHE16300135-1 TMK100KE enzyme included 100 assays $634.60
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BHE16300135-2 TMK500KE enzyme included 500 assays $2025.58
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