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| | In vitro Cyclooxygenase Inhibition Assay |
| Description: | Reaction mixtures were prepared in 100 mM Tris HCl buffer, pH 8.0 containing 1 µM heme and COX-1 or COX-2 and preincubated for 10 min in a waterbath (37 C). The reaction was initiated by the addition of 10 mM arachidonic acid (final concentration in reaction mixture 100 µM). After 2 min, the reaction was terminated by adding 1 M HCl and PGE was quantitated by an ELISA method. The test compounds were dissolved in DMSO and diluted to the desired concentrations with 100 mM potassium phosphate buffer (pH 7.4). Following transfer to a 96-well plate coated with mouse anti-rabbit IgG, the tracer prostaglandin acetylcholine esterase and primary antibody (mouse anti PGE2) were added. Plates were then incubated at room temperature overnight, reaction mixtures were removed and wells were washed with 10 mM potassium phosphate buffer containing 0.05% Tween 20. Ellman s reagent (200 µM) was added to each well and the plate was incubated at room temperature (exclusion of light) for 60 min, unt |
| Affinity data for this assay | |
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