I am working at department of biophysics, Panjab University, Chandigarh, India as a senior Research Fellow. I had an opportunity to use and analyze the COX activity Assay Kit from Cayman Chemical. As part of my work, I was in search of a convenient and efficient protocol for evaluation of COX 1 and COX 2 activity as I am working on phytochemals in the area of cancer research. Cyclooxygenase is a bifunctional enzyme exhibiting both cyclooxygenase and peroxidase activities. The cyclooxygenase component converts arachidonic acid to a hydroperoxy endoperoxide and the peroxidase component reduces the endperoxide to the corresponding alcohol, the precursor of prostaglandins , thromboxanes and prostacyclins. Further, it is now well established that COX 2 is responsible for biosynthesis of prostaglandins during acute inflammatory response. I tested and evaluated various protocols. I rated them with respect to sensitivity and specificity as well as other aspects such as ease-of-use, hands-on time, the level of complexity of results interpretation and the required skill level of the user.
The chemistry utilized in the Cox activity Assay Kit involves monitoring the appearance of oxidized N N N N tetramethyl-p-phenylenediamine (TMPD) at 590 nm which in turn gives the measure of peroxidise activity of COX. Further, the kit also contains isozyme-specific inhibitors for distinguishing COX 2 and COX 1 activity.
I found the Cayman COX Activity Assay Kit instructions to be clear, concise and detailed enough so that anyone skilled in the sciences could perform the assay. Secondly, in the same kit, it is very easy to measure activity of both COX 1 and COX 2 with help of specific inhibitors. Also, the protocol is sensitive enough to allow estimation in a small sample size. The drawbacks of the COX Activity Assay Kit are its cost and that you cannot store the kit at the temperature above -80°C.
The kit allows the user to work on cell lines as well as tissue homogenates. My research work involves use of tissue homogenates. So, prior to dissection, lung tissues were perfused with Tris Buffer pH 7.4 to remove any red blood contamination. Tissue homogenates were made and then centrifuged at 10,000 rpm for 15 minutes at 4°C. The resultant supernatant was used in the assay. For the wells to be used as background/reference wells, an inactive form of all samples was made by taking 150 ul of each sample and boiling for five minutes, followed by centrifugation at 8,000 rpm for one minute. 40 ul of this inactive form of sample was transferred to the background wells of the plate and similarly, active samples were transferred to the sample wells of the plate. COX standard wells involved the transfer of 10 ul of COX standard instead of sample. Assay buffer and Heme was then added to all of the wells. For the determination of COX 1 and Cox 2 activity, the respective inhibitor was also added to the appropriate set of sample wells. The plate was then incubated with shaking at 25°C for 5 minutes. 20 ul colorimetric substrate was then added to each well followed by addition of archiodoinic acid to initiate the reaction. The plate was again incubated with shaking at 25°C. The absorbance was read at 590 nm using a plate reader.
I recommend the COX Activity Assay Kit for its technology, reproducibility, sensitivity, and specificity. The instructions were clear, precise and accurate. In addition, there was very little clean up and preparation time.
Department of Biophysics
Panjab University