| Description | The Nitric Oxide Detection Kit is designed to quantitatively measure Nitrate and Nitrite present in a variety of samples. Nitric Oxide content is derived from the sum of Nitrate (-NO3) and Nitrite (-NO2). Both Nitrate and Nitrite standards are provided to generate standard curves for the assay and all samples should be read off the appropriate standard curve. For Nitrite detection, samples are mixed with the Color Reagents A and B and incubated at room temperature for 5 minutes. The colored product is read at 550 - 570 nm. The concentration of Nitrite in the sample is calculated, after making a suitable correction for any dilution of the sample, using software available with most plate readers. Total Nitric Oxide content is measured after the sample is incubated with Nitrate Reductase and NADH. The reductase in combination with NADH reduces Nitrate to Nitrite. After a 20 minute incubation at room temperature, Color Reagents A and B are added and incubated at room temperature for 5 minutes. The colored product is read and calculated as with the Nitrite determination above. The concentration of Nitrate in the sample is calculated by subtracting the measured Nitrite concentration from the Total Nitric Oxide concentration for the sample. Indirect Quantitative Assay. Intra Assay Precision: Three samples were further diluted in Assay Buffer and run in replicates of 20 in an assay. The mean and precision of the calculated Nitrite or Total NO concentrations were: Sample 1 (Nitrite Concentration)- 45.1 μM, 4.4% CV, Sample 1 (Total NO Concentration)- 70.5 μM, 6.8% CV Sample 2 (Nitrite Concentration)- 73.3 μM, 9.1% CV, Sample 1 (Total NO Concentration)- 107.4 μM, 4.4% CV Sample 3 (Nitrite Concentration)- 132.7 μM, 1.3% CV, Sample 1 (Total NO Concentration)- 157.8 μM, 1.8% CV Inter Assay Precision: Three samples were further diluted in Assay Buffer and run in duplicates in twenty assays run over multiple days by three operators. The mean and precision of the calculated Nitrite or Total NO concentrations were: Sample 1 (Nitrite Concentration)- 44.1 μM, 3.1% CV, Sample 1 (Total NO Concentration)- 68.8 μM, 7.4% CV Sample 2 (Nitrite Concentration)- 66.4 μM, 4.0% CV, Sample 1 (Total NO Concentration)- 112.1 μM, 5.7% CV Sample 3 (Nitrite Concentration)- 126.7 μM, 6.3% CV, Sample 1 (Total NO Concentration)- 154.48 μM, 4.1% CV. Range: 3.125 - 200 μM. Sensitivity: 2.63 μM in the Nitrite and 1.02 μM in the Total Nitric Oxide. Time: 30 minutes. |
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