The NWLSS™ Nitric Oxide Non-enzymatic Assay kit is a system for measurement of nitric oxide as total nitrate and total nitrite in biological samples. This version is best suited for more complex biological samples with higher levels of proteins. Note: This assay requires an overnight incubation to effectively reduce sample nitrates.
Nitric Oxide (NO) is a biologically relevant free radical and cell signaling molecule. In normal cellular physiology it was originally characterized as an endothelial relaxation factor (EDRF) and is most commonly associated with its role in regulation of vasodilation. From a pathogenic standpoint the free radical nitric oxide (NO•) is known to combine with superoxide (O2•) to form peroxynitrite (ONOO-) which is a potent oxidant. Since ONOO– (NO3-) is essentially an unstable isomer of nitrate, it can react with tyrosine residues in proteins to create nitrotyrosine, a biomarker of oxidative mediated modification of proteins.
Nitric oxide degrades rapidly to nitrate (NO3) and nitrite (NO2) in aqueous biological systems. When NO3 is reduced to NO2 measurement of nitrite provides a simple, indirect means to quantify nitric oxide produced in a wide array of experimental model systems.
The test method is based on measurement of total sample nitrites using Griess Reagent. Since Griess reagent does not detect nitrate NO3, This kit employs metallic cadmium for reduction of nitrate to nitrite (Fig. 1). The advantage of using cadmium instead of the enzyme Nitrate Reductase is that sample reduction is not affected by acidic reagents used to precipitate proteins prior to assay.
View Assay Protocol
Quantifying nitric oxide (NO) in biological samples. This is often the preferred method when testing samples with higher levels of proteins.
Plasma, cell lystaes, tissue homogenates
Nitrites or total nitrate & nitrite after nitrate reduction.
|Storage and Stability:||
9 months from date of manufacture when stored at 2-8C
Microplate (96 well clear, low binding, flatbottom)|
Nitrite Standard (500 µM NaNO2)
ZnSO4 (30% wt./vol.)
Nitrate Reductase (Lyophilized)
Reagent A: (Sulfanilamide (p-Aminobenzenesulfonamide) in 3N HCl)
Reagent B: (N-(1-Naphthyl) ethylenediamine dihydrochloride in deionized H2O)
Granulated Cadmium (cadmium (Cd++) beads)
Acid Wash Solution (0.1 M HCl)
Basic Wash Solution (0.1M NH4OH)