Nitric Oxide (NO) Non-Enzymatic Assay Kit, BioAssay(TM)

The Nitric Oxide (NO) Non-Enzymatic Assay Kit allows you to measure total nitric oxide (NO) produced in in vitro experimental systems following conversion of nitrate to nitrite by metallic cadmium. 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 involves conversion of nitrate to nitrite catalyzed by cadmium, followed by quantitation of nitrite using Griess Reagent.\n\nThis kit is suitable for the quantitative determination of total nitric oxide in samples that have high protein concentrations. The cadmium catalyst proves far more robust when in the presence of harsh deproteinating reagents than the alternative, nitrate reductase. \n\nIn addition to providing all necessary components in a microtiter format, this kit employs cadmium beads, which is suitable for rapid, quantitative and inexpensive determination of the total nitric oxide. Nitrate reductase may also be used for this purpose, but cadmium is preferred for highly samples containing relatively large amounts of protein.\n\nPrinciple of the Assay:\nIn aqueous solution, nitric oxide rapidly degrades to nitrate and nitrite (Equations 1 and 2). Spectrophotometric quantitation of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit employs metallic cadmium for quantitative conversion of nitrate to nitrite (Equation 3) prior to quantitation of nitrate using Griess reagent thus providing for accurate determination of total NO production.\n\nNitric Oxide -----------> Nitrate (Equation 1)\n\nNitric Oxide ------------> Nitrite (Equation 2)\n \n Cd\nNitrate -----------------> Nitrite (Equation 3)\n\nThis kit can be used to accurately measure as little as 1pmol/ul (~1uM) NO produced in aqueous solutions. Very little sample is required (1 to 100ul depending on the [NO] in the sample). The completed reaction is read at 540nm.\n\nKit Components:\nN2577-03A: Granulated Cadmium (Cd), 1x25g cadmium beads.\nN2577-03B: ZnSO4 Solution, 30% 1x2ml\nN2577-03C: Microcentrifuge Tubes 50x1.5ml. Use for overnight incubation of the samples.\nN2577-03D: Color Reagent 1, 1x7ml. Sulfanilamide (p-Aminobenzenesulfonamide) dissolved in 3N HCl.\nN2577-03E: Color Reagent 2, 1x7ml. N-(1-Naphthyl) ethylenediamine dihydrochloride dissolved in deionized H2O. Light Sensitive.\nN2577-03F: Nitrite Standard, 1x1.5ml 500pmol/ul NaNO2 (equivalent to 500uM NO).\nN2577-03G: Microtiter Plate, 1x96 wells. Low protein binding plate with flat-bottom wells.\nN2577-03H: Reagent Reservoirs 3 each. For dispensing and pipeting reagents.\nN2577-03J: HCl Washing Solution, 1x125ml. 0.1M HCl\nN2577-03K: NH4OH Washing Solution, 1x125ml. 0.1M NH4OH \n\nStorage and Stability:\nMay be stored at 4 degrees C. Do not freeze. Stable for at least 12 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap. N2577-03E is light sensitive, keep in the dark. Cadmium should be stored in a dedicated dry environment.\n

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