June 2, 2009 - 9:55am
Hello!
I have to measure intracellular reduced equivalents (NADH and FMNH2). I´m going to use a biolumescence method using two coupled-enzymatic reactions (using an oxidorreductase and luciferase).
I have all reagents and with the first tests I´ve no good results. I think that the problem is in one of the enzymes but I´m not sure because I´ve been very meticulous in all the steps. Another possibility is that the quantity of reagents is not correct.
Does someone do the same test?
I´m looking for a kit but it seems that it does not exist...
Thanks!!!!
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The bioluminescent protocol you are trying out has been widely used among others in many applications by Kudryasheva and coworkers, you should definitely check their protocols out (e.g., in Kudryasheva et al (2000) Applied Biochemist and Microbiology, 36, 409-413) to make sure you prepare enzymes and reagents in the proper way regarding concentrations and storage buffers. NADH degrades in solution at room temperature, it needs to be prepared fresh, kept on ice and used within an hour for your assay. You definitely need to generate calibration curves with standard NADH solutions to make sure everything works properly, you could start with a nanomolar to micromolar concentration range.