I've been trying to detect nitrite from bacterial cells cultures in which the bacteria had a construct expressing bNOS. Tried with a commercial kit (from sigma. This one also converts nitrate to nitrite, so I could measure both) but it failed miserably (lots of negative values for my samples. The standards were ok). Thought it was my construct, so I tried again with a different one. Nothing. Then simply tried measuring nitrite with the home made Griess reagents (which I know works because it's what I used to determine if macrophages are activated, and it works perfectly there). Thought that maybe the levels were too low, so changed to using the DAN reagent (fluorometric). Measured my unknown samples (did different dilution) with exactly the same gain as for the standard but, again, more negative values, including for my positive control (MRSA which has a bNOS and should be producing NO, which will be converted to nitrate and nitrite). I did this with 3 different constructs, but every time I get negative concentrations of nitrite. I've thought about the LB media interfering with the assay, but I make my standards with same volume of media I'll be using in the assay, and the standard works. I know it is possible that none of the constructs is actually functional, even though at least one I know for sure it is expressing and the gene was synthesised (with codon optimisation), which will be incredibly bad luck. Anyone had a similar problem, or know what could be going wrong? Any help/ideas would be appreciated.
Thank you.
It is probably related to the concentration which is below detection limit. I suggest to ameliorate the growth conditions (medium, incubation time,...). Otherwise, change the protocol for nitrite measurement.
Dear Claudia:
Griess method is quite sensitive and it allows to quantify nitrite concentration in nanomolar range.
Are you trying to detect nitrite in crude extracts? If the whole metabolic pathway is induced, then nitrite present in your crude extract could be quickly modify by other enzymes or you could get spontaneous oxidation of nitrite to nitrate (spontaneous chemical reaction in the presence of oxygen).
Good luck!
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