I performed the experiment as specified in papers, like
-mixing extract and sodium nitroprusside and incubate 1 hr at 37 degree C.
- add griess reagent and read absorbance at 546 nm
here the problem is that after mixing the griess reagent the appearance of pink colour is not sufficient to reach the od value 0.5-0.7 . there is fade pink colour. i need to wait a long time (5-6 hours) to get small pink colour. in such case it is hard to compare the control and sample groups.
when i prepared sodium nitroprusside solution 2 days before and i used that solution then i get dark pink colour as soon as i add griess reagent. So what do you want to say for it? Shall i not use freshly prepared sodium nitroprusside solution? Please share your views. thank you.
`Hi, I am not sure about your title.
Griess assay measures nitrite (a product of NO oxidation by oxygen), and nitroprusside is an NO donor (or chemical NO generation system).
There's no scavenger per se in the description you provided.
There are many commercial NO donors, and they have different (or very different) half-lives. SNP supposedly has very short half life.
https://books.google.com.hk/books?id=7unHBAAAQBAJ&pg=PA97&lpg=PA97&dq=sodium+nitroprusside+half-life&source=bl&ots=5S2YgFUq3M&sig=mt1aWZlGrdpKJnE2QKexlDZE-8Y&hl=en&sa=X&ved=0ahUKEwin-eGltefJAhUhKKYKHSI2BawQ6AEITjAJ#v=onepage&q=sodium%20nitroprusside%20half-life&f=false
But then, your assay in fact has a lag stage to wait for nitrite to form.
Oxygen supply may affect the efficiency of nitrite formation.
@Nai-Kei Wong thank you for your response. i tried to open the link you shared with me but here it is not accessible. would you please send me the pic of the content . thank you
Respected brother,
Gries reagents used to measure nitrite, automatically generate pink color with the passage of time, so we should measure the nitrite release within short time after mixing with it. As much I know, we should treat the extract to a particular cell that get activated and generate the NO production with particular toxins stimulation, toxins might be like LPS. After 24 hr of treatment, the treated cells supernatant should mixed with Gries reagent in equal proportion, that mixture generate the pink color instantly. That we can measure with 570 nm using microplate reader.
thanks lalita for your reply.... may i know if you use sodium nitroprusside for measuring NO scavenging test? if you use, then i request you to let me know your process.
Sorry brother,
I am not using sodium nitroprusside . I am using Gries reagents containing Sulphonylamide and Naphthy-ethylene-diamine-di-hydrochloride for the measurement of NO produced by LPS activated microglia.
I used the Griess method based on the reaction of NaNO2 with sulfonyl acid, the product absorbs at 540 nm. The values were referred to a standard curve of NaNO2.
This resulted well for murine ovarian homogenates.
This reference could be useful:
https://www.researchgate.net/publication/20016047_Ding_AH_Nathan_CF_Stuehr_DJ_Release_of_reactive_nitrogen_intermediates_and_reactive_oxygen_intermediates_from_mouse_peritoneal_macrophages_Comparison_of_activating_cytokines_and_evidence_for_independe
Regards
Article Release of reactive nitrogen intermediates and reactive oxyg...
thanks carolina for your reply...but my queries are related to the use of sodium nitroprusside.
Dear Ramakanta,
did you manage to solve your problem? Because I am using SNP for the experiment and I encountered your exact problem, my absobances are extremely low.
Please let me know
Ariadna
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