Please help me to solve the problem of my experiment that nitrite oxide scavenging activity assay by using the Griess reagent and sodium nitroprusside.
I get data from my experiment that absorbances increase with the increase of the concentrations (including ascorbic acid as a positive control). I think the pink color should decrease when the concentration increases. But I get opposite results.
I have followed a protocol from an article:
-1 ml extracts (dissolved in PBS) + 1 ml sodium nitroprusside (10mM in phosphate buffered saline (pH 7.4)
-incubation at 37° C for 24h.
-1:1 mixing of sample and Griees reagent for 15 min at dark.
-540 nm.
please share with me the procedure you follow so that I can help you . I have face the same experience and no paper given us the correct procedure.
Upamalika Samanthi I have followed these 3 protocols:
1- To make an aqueous solution of SNP, 10 mM SNP was dissolved in 0.1 M phosphate buffer (pH 7.4). To test the samples, 1 mL of sample dissolved in 0.1 M phosphate buffer was added to a test tubes containing 1.0 mL of SNP solution (10 mM), then incubated at 37o C in a water bath for 24 h. Samples were stirred with a vortex mixer. after incubation and 100 µL of samples were added to 100 µL of Griess reagent (a mixture of 1% sulfanilamide in 5% phosphoric acid, and 0.1% N-(1-naphthyl)-ethylene diamine hydrochloride in distilled water) in a 96-well microplate. The absorbance of samples was measured after 15 min at 540 nm. (Jin, H.J., Lee, J.H., Kim, D.H. et al. Food Sci Biotechnol (2015) 24: 1555. https://doi.org/10.1007/s10068-015-0200-2)
2- Plant extract was dissolved in distilled water for this quantification. Sodium Nitroprusside (5mM) in standard phosphate buffer saline (0.025m, pH 7.4) was incubated with different concentration (100-400µg/ml) of methanol extract and tubes were incubated at 29oC for 3 hours. Control experiment without the test compounds but with equivalent amount of buffer was conducted in an identical manner. After 3 hours incubated samples were diluted with 1 ml of Griess reagents. The absorbance was observed at 550nm.( Singh, D., Mishra, M., Gupta, M., Singh, P., Gupta, A., & Nema, R. (2012). Nitric Oxide radical scavenging assay of bioactive compounds present in methanol Extract of Centella asiatica. Int J Pharm Pharm Sci Res, 2(3), 42-44
3- Sodium nitroprusside (10 mM), in phosphate-buffered saline, was mixed with different concentrations of CS extract dissolved in water and incubated at room temperature for 150 min. Griess reagent (0.5 ml ), containing 1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, was added to the mixture after incubation time. The absorbance of the chromophore formed was read at 540 nm.
Ebrahimzadeh, M. A., Pourmorad, F., & Hafezı, S. (2008). Antioxidant activities of Iranian corn silk. Turkish Journal of biology, 32(1).
Thank you for your consideration.
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