NO donors are molecules that generate or release NO under a given set of conditions, for example NONOates at neutral or acidic pH.
NO scavengers are molecules with high affinity for NO that remove or destroy it, for exmaple hemoglobin.
Thanks peter sobolewski, will you please provide me procedure of griess assay for Nitric oxide donor activity, ----- [email protected]
The basic Griess assay for nitrite (the resultant of NO decomposition) is very simple.
You will need:
sulfanilamide (Sulf)
N-1-napthylethylenediamine dihydrochloride (NED)
Phosphoric acid
Sodium Nitrite to prepare standards
(You should have no issue getting any of these reagents from Sigma or whatever chemical supplier you use or you can look for a kit, but frankly that's a waste of money.)
Prepare fresh each time:
1% Sulf in 5% phosphoric acid
0.1% NED in water
Easiest is to use a 96-well plate:
Add 50uL of sample, followed by 50uL of Sulf solution.
Incubate for 10 min (or so) in the dark.
Add 50uL of NED solution
Incubate for 10 min (or so) in the dark -- you should see pink/purple color developing in your positive samples.
Measure ABS at 520 or 530 (or 540) depending what filters/etc you have.
Make sure you have a blank that is the solvent/buffer/media your samples are in.
Make sure you run a standard curve via serial dilution from say 1 to 100uM nitrite.
For other formats (cuvettes, etc) just adjust the volumes while preserving the 1:1:1 ration.
In principle you could just combine it all at once in one step, but in my experience this serial method is more effective, and it's not really a hassle. Also, you could reuse the stocks over the course of a few weeks if you store them in the fridge, but preparing fresh is best and again, not much of a hassle.
Thanks for reply, it is helpful to me while performing experiments i will contact you, for advice.. can u provide your email address.
I think it's best to post additional questions/comments here, so others can also benefit from the exchange, rather then keeping it private -- there are several "threads" regarding NO, Griess, etc. so this may add to the knowledge base.
However, if you do need to send me a personal message, please use the messaging system built into RG, via my profile.
@ Peter... ok i will use research gate regularly, not issue to keep private. regards Swami Prabhu
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