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| | COX Inhibitor Screening Assay |
| Description: | Dilute 3 mL of assay buffer concentrate with 27 mL of HPLC - grade water. This final assay buffer (0.1 M Tris-HCl, pH 8) should be used for dilution of heme and COX enzymes prior to assaying. This vial contains a solution of heme in dimethylsulphoxide. Dilute 88 μL of heme with 1.912 mL of diluted assay buffer prior to use. A vial contains a solution of ovine COX-1 and should be kept on ice when thawed. Dilute 200 μL of enzyme with 400 μL of diluted assay buffer and store on ice. A vial contains a solution of ovine COX-2 and should be kept on ice when thawed. Dilute 200 μL of enzyme with 400 μL of diluted assay buffer and store on ice. A vial contains a solution or arachidonic acid in ethanol. Transfer 100 μL of the supplied substrate to another vial, add 100 μL of potassium hydroxide (item no. 760115), vortex, and dilute with 1.8 mL of HPLC - grade water to achieve a final concentration of 1.1 mM. Use the prepared arachidonic acid solution within 30 min. A 20 μL aliquot will yield a final concentration of 100 μM in the wells. A vial contains 0.1 M potassium hydroxide (KOH). A vial contains a solution of TMPD. Background wells: add 160 μL of assay buffer, and 10 μL of heme to three wells. 100% Initial Activity wells: add 150 μL assay buffer, 10 μL of heme, and 10 μL of enzyme (either COX-1 or COX-2) to three wells. Inhibitor wells: add 150 μL of assay buffer, 10 μL of heme, and10 μL of enzyme (either COX-1 or COX-2) to three wells. Add 10 μL of inhibitor to the inhibitor wells and 10 μL of solvent (methanol, dimethylsulphoxide or ethanol) to the 100% Initial Activity wells and background wells, this process was repeated three times. Carefully shake the plate for a few seconds and incubate for five minutes at 25 °C. Add 20 μL of the colorimetric substratesolution to all the wells that you are using. Add 20 μL of arachidonic acid to all the wells you are using. Carefully shake the plate for few seconds and incubate for five minutes at 25 °C. Read the absorbance at 590 nm using a plate reader. |
| Affinity data for this assay | |
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