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Thanks you very much for your help! After I talked to you on the phone, I went back and tried another dilution series with one of my original plasma samples (up to 1:64 dilution). I used both kinetics and absorbance at 450nm after stop solution was added. A spreadsheet with results from both tests is attached. I would very appreciate if you could take a look at them and maybe give me some suggestions what I should do next!
Asked on Sep 07 2012
Thank you for providing this information.
I have reviewed this with my colleagues in the lab. It appears to us as if the very high protein concentrations are affecting the assay. I can’t understand why it would be a negative curve though unless there is some antibody precipitation or pH effect.When looking at the lower concentrations 10ug/mL and 1ug/mL this is not seen.Following this is seems the best action would be to use less protein sample and make sure that you are using exactly the same dilution buffer for all samples and standards to avoid any minor effects from interference or differences in concentration.
When reviewing the lower concentration sample in your spreadsheet it looks as there is not a significant level of nitration in the sample at these concentrations.
FYI peroxynitrate can be obtained commercially from EMD Millipore; this can be added to samples to act as another positive control which would completely quench the signal.
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Answered on Sep 07 2012