I'm testing for NO assay, using Griess reagent. As stated in the protocol, I did serial dilution of the standard. Upon adding both reagents, the OD obtained in standards (with known concentration) will be compared with the samples with unkown NO concentration. Weird enough, my samples did not emit the same colour intensity as the standards, but the OD readings were almost the same as the standard. why is this happening? can you help me explaining this issue? Thanks in advanced!
Dear Fairuz,
As you know, this test is for the determination of nitrite via forming a purple color when the reagent is added to the compound.
It seems that your compound has some color. Upon adding the reagent to a colored compound the resulting color is expected to be slightly different from the pink color obtained when using the standard.
Despite this fact, at the reading lamda, the complex formed between your compound and the reagent has absorption as that for the complex of the standard and the reagent.
Hoping this will be helpful,
Rafik
Dear Fairuz, I agree with Rafik Karaman, check your buffer that was used for samples with unknown NO concentration. There (in that buffer) might be something (for example, a little bit of culture medium left, or something like this) that affects the sample colour in the NO assay. Probably, if you will solve this issue, everything will be alright!
Success!
agree with both Rafik and Marina, make sure that you buffer composition is compatible with Greiss reagent, check for detergents, denaturing agents, typically they interfere with chromophores
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