Department of Biophysics
Panjab University
As part of my work, I was in a search of a highly specific and sensitive protocol for the evaluation of NOS (nitric oxide synthase) activity as I am working on phytochemicals in the area of cancer research and I wanted to analyze the effect of a combined treatment of phytochemicals on the activity of NOS. NOS is a key enzyme which plays a central role in the production of the cellular messenger NO (nitric oxide) from arginine. 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 principle of the NOS Activity Assay Kit from Cayman Chemical is the monitoring of the conversion of arginine into citrulline using radioactive substrates. Radioactive arginine is added to protein extract (if the sample is from tissue) or to purified NOS samples (if the sample is from a cell line). After incubation, the reaction is stopped with an EDTA-containing buffer, which chelates the calcium required by the NOS enzyme, which finally leads to the inactivation of the enzyme. Arginine-binding resin is then added to the sample and the entire reaction is pipetted into spin cups. The citrulline flows through the cups and NOS activity is then quantified by counting the radioactivity in eluate.
I found the Cayman NOS Activity Assay Kit instructions to be clear and comprehensive enough that anyone skilled in basic bench technique could perform the assay. Another thing I liked about this kit is that you can work with samples from either tissue or cell lines. Also, the protocol is sensitive enough to allow estimation in the picomole range. The drawbacks of the assay kit are its cost and use of radioactive substrates. Also, a liquid scintillation counter is a must.
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 followed by centrifugation at 10.000 rpm for 15 minutes at 4°C. The resultant supernatant was used for the assay. Then, as directed in the kit instructions, different reagents such as the reaction buffer, stop buffer, calmoduline, equilibrated resin and calcium chloride solutions were prepared. Accordingly, 250 µl of reaction buffer, 50 µl of NADPH, 10 µl of 3H-Arginine, 50 µl of calcium chloride and 40 µl of HPLC grade water were pipetted into the microcentrifuge tube. I took 40 µl of this reaction mixture and transferred it to another microcentrifuge tube, added 5 µl of calmodulin and 5 µl of sample. The microcentrifuge tube was then incubated at 22-37°C for 60 minutes. I stopped the reaction by adding 400 µl of stop buffer. I then added 100 µl of equilibrated resin into the sample tube and transferred everything to a spin cup. This was followed by centrifugation for 30 sec and then eluates were measured on a liquid scintillation counter.
The results I obtained were highly reproducible as we often repeat a particular experiment twice and take the readings in triplets. So as to check the reproducibility, we ran experiments with this kit three times and found readings to be very stable with minimal variation, The statistical significance of the results were of level of 0.001 analyzed by statics software SPSS 14, using ANOVA statistic test. The kit also ranked good in terms of sensitivity and specificity as it can allow estimation of the samples in the picomole range. Further, the instruction manual and reagents provided to run an experiment make the kit very easy to use. As far as interpretation of the results are concerned, the kit also provides assistance with calculations by providing easy equations.
I recommend the NOS activity Assay Kit for its specificity, sensitivity and reproducibility. The instructions were clear and comprehensive.