Pricing updated 2019-07-21. Prices are subject to change without notice.
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.
Warning - this product is not for human or veterinary use.
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Matouk, A.I., Taye, A., El-
Ishimoto, Y., Inagi, R., Yoshihara, D., et al. Mitochondrial abnormality facilitates cyst formation in autosomal dominant polycystic kidney disease Mol. Cell. Biol. 37(24), e00337-00317 (2017).
Datta, K., Suman, S., Kallakury, B.V., et al. Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine PLoS One 7(8), e42224 (2012).
What are the main differences between the Nitrate/Nitrite Colorimetric Assay Kit and Nitrate/Nitrite Colorimetric Assay Kit (LDH method)?
The Nitrate/Nitrite Colorimetric Assay , product number 780001, uses a NADPH cofactor recycling method which allows use of a NADPH concentration that is below the level of interference in the Griess reaction. The Nitrate/Nitrite Colorimetric Assay (LDH Method), product number 790871 uses LDH to eliminate all excess NADPH that is required for NOS activity assays. The LDH kit also removes excess NADPH used to catalyze conversion of Nitrate to Nitrite. Of the two nitrate/nitrite assays mentioned, we recommend the Nitrate/Nitrite Colorimetric Assay (or the corresponding Fluorometric assay if greater sensitivity is needed) for most applications since it is significantly easier to perform. The only reason to use the Nitrate/Nitrite Colorimetric Assay Kit (LDH method) is for measurement of Nitrate/nitrite from in vitro NOS activity assays.