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Assay Method Information

Assay Name:  In vitro COX Inhibition Assay
Description:  COX fluorescent inhibitor screening assay kit (catalog number 700100, Cayman chemical, Ann Arbour, MI, USA) has been employed to investigate the isozymes specifity of some of the synthesized compounds 7a, 7b, 8c, 8e, and 13 following the procedure suggested by manufacturer. Briefly, in 96-well plate, either ovine COX-1 or human re-combinant COX-2 has been incubated with different concentrations of each tested compound in presence of the assay buffer (100 mM Tris-HCl, pH 8.0), heme and the fluorometric substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) for 20 min at room temperature. The reaction started after addition of arachidonic acid solution for 2 min at room temperature. Fluorescence of resorufin that is produced by the reaction of between PGG2 and the fluorometric substrate were analysed. Dup-697 (selective COX-2 inhibitor) and SC-560 (selective COX-1 inhibitor) were used as referencecompounds. The measured fluorescence intensity is proportional to the amount of PGG2 present in each well during the reaction. IC50 (μM) that corresponds to the concentration of the inhibitor that causes 50% inhibition of COX-1 or COX-2 activity was calculated from the dose response curve of inhibition (triplicate determinations).
Affinity data for this assay
 

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Last update November 1, 2007
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