The common method for assessing eNOS function requires addition of radioactive L-arginine, however, I can not use this method, are there other methods available that I can use. Can I perhaps induce NO production using a cytokine like IL-1beta or non-radioactive arginine, will the results reflect on the functionality of the enzyme?
You're conflating a few different assays here.
First, the radiolabeled Arg assay is a classic NOS activity assay, assessing the conversion of Arg to Cit. It's done in enzyme extracts with addition of all needed cofactors, etc.
The Griess assay, on the other hand, measures nitrite (and nitrate, if you use a reductase to convert it to nitrite). Nitrite is an aqueous oxidation product of NO, and can be considered to be an indicator of NO bioavailability. This assay can be done with cultured cells -- sample the media -- but has limited sensitivity. Depending on your cells and culture conditions (cell number, volume of supernatant, etc.), it can work, given the right stimulus. You'll need to provide some more info.
As Peter and Subhadeep explain, the Griess assay (with reduction of nitrate to nitrite step) is simply in aindication of global NOS activity/function. Obviously, if there is an abundance of eNOS compared to iNOS, one may infer that NO release as measured by griess assay may be a reasonable indicator. However, i suggest to buy probes for the 3 NOS isoforms and peform RT-PCR.
Thank you all for your answers. I am attempting a quick assay to assess eNOS in rat heart tissue and compare different samples and I have Griess reagent which measures NO (after its conversion to nitrate) as does the dye DAF-2DA etc.I have managed to separate the membrane bound eNOS from the free enzyme iNOS however I think RT-PCR might still be necessary. I was thinking of trying to simulate eNOS which might be difficult considering it is constitutively expressed and am looking for suggestions. After stimulation I could then compare my samples based on product formed (NO production based on the Griess assay) and not substrate used which is the case with the classic NOS activity assay. Alternatively I could add L-arginine ( non-radioactive) and co-factors and measure how much of NO (using Griess assay) is actually formed. I have been looking for a method like this in literature but have not found any yet and I am trying to find out if anyone has perhaps tried alternative methods which do not require a kit, and what could have been a flaw in the experimentation.
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