NADPH is an essential cofactor for the function of the NOS enzyme. Unfortunately, NADPH interferes with the chemistry of the Griess reagents, which are the most commonly used reagents for nitrite detection. There are two ways to get around this limitation. Vanishingly small amounts of NADPH can be used, in conjunction with a catalytic system for recycling the spent NADP+ back to NADPH. This is the system used in Cayman’s first Nitrate/Nitrite Colorimetric Assay Kit (Item No. 780001). It works well for the analysis of fluids such as plasma and urine, and is also available in a high sensitivity fluorometric version (Item No. 780051) for the detection of low levels of nitrate and nitrite. However, this method cannot be used to analyze nitrate and nitrite from an in vitro NOS assay in which excess NADPH has been added. The second way to handle the NADPH interference problem is to go ahead and use an excess of NADPH. An additional step is added to the protocol to remove the excess NADPH. This is the avenue we have taken in the development of this nitrate/nitrite assay kit. This kit uses Lactate Dehydrogenase (LDH) to oxidize the excess NADPH. The kit is particularly well suited to measurements of NOS activity in vitro. High throughput screening applications using recombinant nitric oxide synthase (NOS) preparations can be quickly and accurately assayed with the LDH method kit. Each kit contains assay buffer, nitrate reductase, cofactors preparation, nitrate standard, lactate dehydrogenase, Griess Reagent R1, Griess Reagent R2, a 96 well plate, and complete instructions.Needed but not supplied: Please download the kit booklet to verify if UltraPure Water (Milli-Q or equivalent) or any other components are needed for this assay.WARNING This product is not for human or veterinary use.
