I want to perform de Griess assay without cells, therefore I have used Sodium Nitroprusside (SNP) which in PBS forms espontanously NO.
I have added 50 uL in a 96-well plate of SNP 10mM and added 50 uL if my sample (final conc. SNP=5 mM). I have let this incubate for 180 min at RT.
Then I have added the Griess reagent, in two steps, first the sulphanilamide (50 uL, 1%) in 2.5% phosphoric acid, incubate 5 min, and then I have added the NDP (I have used sigma aldrichs option: Griess reagent modified), also dissolved in 2.5 phosphoric acid, concentration 0.1%.
My problem is that I see colour, but very faint, and when I measure absorbances they are really low, around 0.08 the highest.
What am I doing wrong? I think I have to make concentrations up, but the griess reagent is always this exact concentration I used. I can also make SNP concentration up, but then I should see more colour on the wells without SNP, and containing the standard of NaNO2, and they are also faint.
Any suggestions?
Thanks!
1. You're not extracting the NO from your sample matrix. It has to be acidic.
2. Why are you expecting the NO concentration to be higher?
3. Did you check how to make up the SNP reagent in the APHA's 'Standard Methods of Water and Wastewater'?
Your reagents sound fine.
What ratio of reagents are you using? You don't say how much of the NED (the actual azo dye)
For example I would typically use 50 uL of sample/standard, 50 uL Sulph, wait 5 min, then 50 uL NED, then incubate for 10-15 min at RT--you could always go longer, just protect everything from light.
What wavelength are you using for the absorbance measurement? You want to be in the range of 530 or 540 nm.
Have you done a standard curve of nitrite? Like 1-100 uM?
You should see strong color change by eye in your standard curve.
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