The Analyte:This kit employs immunoaffinity purified Nitrate Reductase to allow you to measure total nitric oxide (NO) produced in in vitro experimental systems following enzymatic conversion to nitrite.
Nitric oxide can be spectrophotometrically assayed by measuring the accumulation of its stable degradation products, nitrate and nitrite. The ratio of these two products in biological fluids, tissue culture media, etc. may vary substantially. Hence, for accurate assessment of the total nitric oxide generated, one must monitor both nitrate and nitrite. An excellent solution to this problem is the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase (NaR), followed by quantitation of nitrite using Griess Reagent.
In addition to providing all necessary components in a microtiter format, this kit employs affinity purified nitrate reductase and NADH, thereby circumventing some of the potential problems reported for nitric oxide measurement using NADPH dependent nitrate reductases.
Principle of the Assay:In aqueous solution, nitric oxide rapidly degrades to nitrate and nitrite. Spectrophotometric quantitation of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit employs the NADH-dependent enzyme nitrate reductase (NaR) for quantitative conversion of nitrate to nitrite prior to quantitation of nitrate using Griess reagent — thus providing for accurate determination of total NO production
This kit can be used to accurately measure as little as 1pmol/ul (~1uM) NO produced in aqueous solutions. Very little sample is required (5 to 85ul depending on the [NO] in the sample). The completed reaction is read at 540nm.
Storage and Stability:*Store N2577-02A at -20°C. **Store N2577-02D powder at RT, liquid at -20°C; Light Sensitive. Store other components at 4°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
