Nitric Oxide Colorimetric Detection Kit

The Nitric Oxide Colorimetric Detection Kit is designed to detect and quantify the level of nitric oxide (NO) in various samples. The physical properties of NO make direct detection methods challenging, but colorimetric methods are effective for measuring its stable break-down products: nitrate (–NO₃) and nitrite (–NO₂).

This assay measures the level of nitrates and nitrites in serum, plasma, urine, saliva, water, buffered solutions (Tris, HEPES, or PBS at pH 7.2; EDTA at ≤10 mM), cell and tissue lysates, or tissue culture medium. The assay was characterized with human samples, but can be used to test samples from other species.

This complete, ready-to-use kit includes two clear 96-well plates, nitrate and nitrite standard, assay buffer, and other components to perform the assay. A 96-well microplate reader capable of reading optical density at 540–570 nm is required for use of this kit.

Performance characteristics
• Assay type: colorimetric detection kit
• Sample types: serum, plasma, urine, saliva, water, buffered solutions (Tris, HEPES, or PBS at pH 7.2; EDTA at ≤10 mM), cell and tissue lysates, or tissue culture medium
• Sensitivity: 2.63 μM nitrite, 1.02 μM total NO
• Standard curve range: 3.125–200 μM
• Reactivity: species independent

Assay principle
The Nitric Oxide Colorimetric 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 color reagents A and B and incubated at room temperature for five minutes. The colored product is read at 540–570 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 five 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.

This kit uses nitrate and nitrite standard solutions calibrated to the US National Institute for Science and Technology Standard Reference Materials and ISO/IEC standards.