The NWLSS™ Nitric Oxide Synthase Activity kit is ideally suited for testing NOS activity of cells in culture after washing. Cell lysates and tissue homogenates can also be tested but requires that baseline nitric oxide be tested and subtracted from timed results.
Nitric oxide synthase (NOS) is involved in the synthesis of nitric oxide from L-arginine. Mammalian NOS include three distinct isoforms; inducible, endothelial and neuronal. The traditional method for measuring nitric oxide synthase (NOS) activity is performed by radiochemical assay that measures the conversion of L-[3H]arginine to L-[3H]citrulline. This method is expensive and requires regulation of radioactive materials.
In contrast, we offer a non-radioactive method that utilizes a simple NADPH recycling system to allow NOS to operate linearly for hours as nitric oxide derived degradation products nitrate and nitrite accumulate. NOS activity can therefore be assayed spectrophotometrically by measuring total nitrite accumulation over time.
NADPH and L-arginine are required for the continual operation of NOS and production of nitric oxide (NO). In aqueous solution, NO rapidly degrades to nitrate and nitrite. Spectrophotometric quantization of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit employs recombinant nitrate reductase (NaR) for conversion of nitrate to nitrite prior to quantization of nitrite using Griess reagent , thus providing for accurate determination of total NOS activity.
View Assay Protocol
oxide (NO) in biological samples
Nitric Oxide as total Nitrite
1 pmol/mL or 1µM in the assay
|Storage and Stability:||
1 year from manufacture date when stored at specified temperature
Microplates 1 X 96 wells|
Reaction Buffer (50 mM HEPES 0.5 mM EDTA 1 X 25 mL
Nitrite Standard (500 µM NaNO2) 1 X 1.5 mL
Nitrate Reductase (Lyophilized) 1 Unit
Nitrate Reductase Buffer 1.2 mL
NADPH Part A (NADP+ , Glucose 6-Phosphate, L-Arginine) 1 mL
NADPH Part B (Glucose 6-Phosphate dehydrogenase) 1 mL
Reagent A: (Sulfanilamide (p-Aminobenzenesulfonamide) in 3N HCl) 1 X 7 mL
Reagent B: (N-(1-Naphthyl) ethylenediamine dihydrochloride in deionized H2O) 1 X 7 mL