I've set up both colorimetric and fluorometric assays for COX-2. I don't remember whether I made them up or derived them from publications. I have not published the methods, but I can share them with you. I've also used a TLC assay to confirm the results of the other assays, since inhibitors can interfere with their readouts.

The necessary reagents are COX-2 enzyme, arachidonic acid, 10-acetyl-3,7-dihydroxyphenoxazine (Ampliflu Red), and hematin.

I think this paper helps

An ELISA method to measure inhibition of the COX enzymes

Nat Protoc. 2006;1(4):1915-21. doi: 10.1038/nprot.2006.308.

Authors

Muriel Cuendet 1 , Andrew D Mesecar, David L DeWitt, John M Pezzuto

Affiliation

1 Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, College of Pharmacy, Nursing and Health Sciences, and The Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.

PMID: 17487176

DOI: 10.1038/nprot.2006.308

Abstract

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).

Cited by 11 articles

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals

Cyclooxygenase 1 / isolation & purification

Cyclooxygenase 2 / isolation & purification

Cyclooxygenase Inhibitors / analysis*

Dinoprostone / analysis

Enzyme-Linked Immunosorbent Assay / methods*

Humans

Sheep

Spodoptera

Substances

Cyclooxygenase Inhibitors

Cyclooxygenase 1

Cyclooxygenase 2

Dinoprostone

Grant support

P01 CA48112/CA/NCI NIH HHS/United States

Full text links

Nature Publishing Group 

@Adam B Shapiro

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