Product Overview :
dCK Assay Kit was specially designed to follow the enzymatic activity of purified deoxycytidine kinase (dCK, EC 2.7.1.74) in vitro. This user manual gives the instructions for standard assays in 96-well plate.
The principle of the assay is based on the use of deoxyinosine (dIR) as a substrate of dCK and a coupled reaction involving a highly active IMPDH (Inosine Monophosphate Dehydrogenase, bacterial recombinant) for a direct measurement of the deoxyinosine monophosphate (dIMP) formed by dCK.
(1) In the presence of dIR and ATP, dCK catalyses the phosphorylation of dIR to form dIMP and ADP.
(2) The dIMP formed is then oxidized to deoxyxanthosine monophosphate (dXMP) by IMPDH in the presence of NAD, leading to NADH2 formation.
This coupling reaction is immediate when IMPDH activity is much higher than dCK activity in the assay. The enzymatic activity of dCK, which corresponds to the formation kinetics of dIMP, is then stoichiometrically and directly monitored by the formation kinetics of NADH2.
The velocity of NADH2 formation is measured with a spectrophotometer at 340nm (molar extinction coefficient of NADH2 at 340nm = 6220 M-1.cm-1).
Description :
Human deoxycytidine kinase enzyme has a broad substrate specificity and provides the phosphorylation of purine/pyrimidine deoxynucleosides and pyrimidine ribonucleosides. Deoxycytidine kinase is critical for phosphorylation of cytidine analogs and provides first steps of activation of highly effective anti-cancer prodrugs, such as aracytidine, gemcitabine and cladribine.
Storage :
dCK Screening Assay Kit must be stored at -20°C until used.
Size :
1 plate (96 assays)
Kit Components :
A standard dCK Screening Assay Kit (one 96-well plate) contains:
1. one tube "IMPDH";
2. one tube "Cofactor 1";
3. one tube "Cofactor 2";
4. one tube "Cofactor 3";
5. one vial "Reaction Buffer 5x";
6. one tube "Deoxyinosine";
7. one tube "Human dCK enzyme", 100mU;
8. Transparent 96-well plate.
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