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).
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Acknowledgements
The authors are grateful to the National Cancer Institute for support provided under the auspices of program project P01 CA48112 entitled “Natural Inhibitors of Carcinogenesis” and the Fogarty International Center for support provided under the auspices of an International Cooperative Biodiversity Group (ICBG) “Studies on Biodiversity of Vietnam and Laos.”
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Cuendet, M., Mesecar, A., DeWitt, D. et al. An ELISA method to measure inhibition of the COX enzymes. Nat Protoc 1, 1915–1921 (2006). https://doi.org/10.1038/nprot.2006.308
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DOI: https://doi.org/10.1038/nprot.2006.308
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