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Phosphorylation of Nucleoside Analogues by UMP-CMP Kinase
Aim: Coupled dCK-CMK nucleoside phosphorylation assay is a cost-effective rapid assay that delivers in one step the critical information on both dCK and CMK substrate properties of nucleoside analogue.
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Assay condition: Substrate properties of nucleoside analogue for dCK and CMK kinases are evaluated in a two-step spectrophotometric assay carried out at 37°C, at 50mM Tris-HCl pH7,6; 50mM KCl, 10mM MgCl2, 5mM ATP, 0.1mM NADH, 1mM phosphoenolpyruvate, 1mM DTT, PK 10U/ml, LDH 15U/ml. The initial phosphorylation is started by addition of dCK (1µM) and the reaction is followed spectrophometrically at 340nm during 90 min followed by addition of CMK (0,3µM). The changes in absorbance at 340nm are used to calculate both the initiale rate of reactions and the concentration of nucleoside monophosphate formed. |
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Method validation: The phosphorylation kinetic of CMP by recombinant CMK have been measured in two independent approaches. In first one, CMK Km for CMP was studied directly with CMP substrate (grey), and in second one CMK Km for CMP was measured indirectly in coupled dCK-CMK assay (red) usine cytidine as a substrate. As shown on left, coupled dCK-CMK assay produces results which are highly similar to those obtained from a direct CMK assay.
A confirmation by HPLC analysis of the formation of phosphorylated forms is available upon request. |
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