Hello,I did My NO estimation using Gries Reagent.I am doing 1ml of total reaction volume,for standard,I have taken 500 microlitre of ddH20and 500 of different conc of Na-nitrite.For my unknown I have taken,800 microlitre of ddH20+100 of G.R and 100 from my unknown sample to make total volume of 1ml. For protein estimation I did Bradford estimation.From my exp. I got the following readings-
For eg.in liver protein conc. is 5.6 microgram per microlitre and For NO is 12.12 microM.From which formula do I have to calculate,Please suggest.
µM=µmole/liter=pmole/µl so in your example you have 12.12pmole/5.6µg or ≈2.16pmole/µg protein.
I hope that helps
12.12 µmoles/liter nitrite in a reaction volume of 0.001 liter = 12.12 x 10-3 µmoles.
5.6 µg protein/µl of sample x 100 µl of sample in reaction = 560 µg of protein in reaction = 0.56 mg.
12.12 x 10-3 µmoles nitrite/0.56 mg = 0.022 µmoles nitrite/mg protein.
To express this as a specific activity, you also need to divide by the time in minutes for the reaction, and also to give all the other conditions of the reaction (pH temperature, buffer, salts, substrate concentration). It is concerning that you said you ran the reaction in distilled water and do not mention any buffer.
hello Sir,I am not using any buffer I am using water,and if I am doing this in wrong way then please suggest me the wright procedure and in case if you have any paper on this then plz send me sir.
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