Product Overview :
Once released outside the cell, adenosine, a naturally occurring ribonucleoside, reveals strong neuroprotective and anti-inflammatory properties. One of the strategies to increase extracellular adenosine consists in inhibiting adenosine kinase (ADK), a ubiquitous enzyme that catalyzes the transfer of γ-phosphate from ATP to the 5'-hydroxyl of adenosine generating AMP and ADP.
ADK Assay Kit is a non isotopic assay that allows a continuous monitoring of ADK activity at 340nm through the coupling of ADK-catalyzed phosphorylation of inosine to the oxidation of IMP by a recombinant IMP-dehydrogenase (IMPDH). The assay developed in microplate format allows a HTS search of novel ADK inhibitors.
ADK Assay Kit was validated with a known ADK inhibitor (A-134974, IC50=20nM) and meets the requirements of a convenient and reliable HTS assay (microplate format, "add-and measure", spectrophotometric continuous readout, Z-factor = 0.68).
ADK Assay Kit can be also used to evaluate the phosphorylation of novel nucleoside analogues. In the absence of nucleoside
competitor, adenosine kinase phosphorylates inosine resulting in the formation of IMP directly monitored by the formation kinetics of NADH2 catalyzed by IMP-dehydrogenase. In the presence of nucleoside competitor, the phosphorylation of inosine, poor ADK substrate, is inhibited and detected as a decrease in NADH2 formation.
Description :
The enzyme adenosine kinase (AdK; EC 2.7.1.20) catalyzes the transfer of gamma-phosphate from Adenosine triphosphate (ATP) to adenosine (Ado) leading to formation of Adenosine monophosphate (AMP). In addition to its well-studied role in controlling the cellular concentration of Ado, AdK also plays an important role in the maintenance of methylation reactions.
Quality Control Test :
Measurement of Z'Factor of ADK assay.
Determine the absorbance rate per minute for every negative controls (ARC-), positive controls (ARC+) and blanks (ARBlank).
Calculate ARC- and ARC+, respectively the means of ARC- measurements and of ARC+ measurements.
Calculate σC- and σC+, respectively the standard deviations of ARC- measurements and of ARC+ measurements.
Z'Factor is then calculated as:
Z' = 1- (3σc++3σc-)/| ARC+ – ARC-|
Z-factor for ADK screening assay = 1-(3*0.0000594+3*0.000064)//0.000523-0.009454/ = 0.958-excellent assay.
Storage :
ADK Assay Kit must be stored at -20°C until used.
Size :
1 plate (96 assays); 5 plates (5 x 96 assays).
Kit Components :
A standard ADK Assay Kit (one 96-well plate) contains:
1. one tube "IMPDH "
2. one tube "Cofactor 1" (DTT);
3. one tube "Cofactor 2" (NAD);
4. one tube "Human recombinant Adenosine Kinase";
5. one tube "ATP";
6. one vial "Reaction Buffer 5x";
7. one tube "ADK inhibitor" (A-134974);
8. one tube "50mM inosine";
9. Transparent 96-well plate.
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