Colorimetric Nitric Oxide Assay Kits

Accurate measurment of Nitric Oxide (NO) in biological fluids and culture media is critical to the assessment of the role of this unique biological mediator in many experimental systems. However, in aqueous solution, NO rapidly degrades to nitrate and nitrite and the ratio of these two products may vary substantially. Although quantitation of nitrite using Griess Reagent is straightforward, it does not measure nitrate. We now offer two kits for the accurate quantitation of NO.

One kit employs immunoaffinity purified NADH-dependent Zea Mays nitrate reductase for quantitative conversion of nitrate to nitrite prior to quantitation of nitrite using Griess reagent - thus providing for accurate determination of total NO production. A second, nonenzymatic kit for NO quantitation employs cadmium beads for the reduction of nitrate to nitrite, and is especially useful for samples containing high protein concentrations - which may interfere with quantitation using the enzymatic kit. The cadmium beads may also be recycled, reducing assay costs, using instructions provided in a convenient refill kit.

Colorimetric Enzymatic NOAssay Kit: This sensitive, convenient and inexpensive kit employs nitrate reductase to allow you to measure total NO produced in in vitro experimental systems following enzymatic conversion to nitrite. This kit can be used to detect generation of less than 1 pg/µl (1 µM) of NO. Each kit includes affinity purified nitrate reductase, NADPH, nitrate standard, color reagents, buffer, an optically clear microtiter plate, and is sufficient for construction of a standard curve and analysis of up to 40 samples in duplicate. Samples ranging from 5 to 85 µL can be used, and the completed reaction is read at 540 nm. Indicator dyes, including phenol red, in tissue culture media do not interefere with this assay. However, it is important to determine the basal nitrate level in the medium used. Complete analysis requires less than one hour following the detailed instructions included with the kit.

Product Number: NB 98 Instruction Booklet-Requires Adobe Acrobat

Colorimetric Non-Enzymatic Nitric Oxide Assay Kit: This sensitive, non-enzymatic and inexpensive kit employs cadmium to allow measurement of total NO produced in vitro experimental systems following non-enzymatic conversion to nitrite. This kit can be used to accurately measure as little as 1pmol/µl (1µM) of NO. Each kit includes cadmium, nitrite standard, color reagents, zinc sulfate solution,and an optically clear microtiter plate. The kit is sufficient for construction of a standard curve and up to 40 samples in duplicate. The complete reaction is read at 540 nm. It is especially useful for measurement of NO in samples with protein concentrations higher than 1mg/well. Indicator dyes in media, including phenol red, does not interfere with the assay.

Product Number: NB 88 Instruction Booklet-Requires Adobe Acrobat

Nitric Oxide Synthase

Inducible Nitric Oxide Synthase

Inducible NOS (iNOS) is a Ca2+-independent isoform that is expressed, upon induction by cytokines, in several cell types including: macrophages, endothelial cells, and hepatocytes.

Murine iNOS Promoter/Enhancer region CAT Construct: This plasmid contains the 5'-flanking region of the murine iNOS gene (nucleotides -1588 to +161 numbered with +1 as the initiation site) linked to a reporter gene which encodes chloramphenicol acetyltransferase (CAT). Upon transfection into a murine cell line, this construct confers inducibility of the iNOS promoter by LPS synergistically with interferon gamma. ( J. Exp. Med. 177:1779-1784, 1993).

Product Number: NS 03

Murine iNOS CMV Promoter Construct: This plasmid contains the iNOS cDNA under the cytomegalovirus (CMV) promoter. This construct can be used for transient expression of mouse iNOS in suitable cell lines such as human renal epithelial cells. ( J. Biol. Chem. 269:28500-28505, 1994).

Product Number: NS 05

Anti-Inducible Nitric Oxide Synthase: This polyclonal antibody, developed against a peptide corresponding to residues 1131-1144 of murine iNOS, is specific for this 130 kDa enzyme in human, rat, and mouse cells. It is useful for immunoprecipitation and Western blotting applications.

Product Number: NS 01

Inducible Nitric Oxide Synthase, Partially Purified: This 100,000 x g supernatant fraction is isolated from stimulated mouse macrophage (RAW cells). Each vial contains 50 units, wherein one unit is the quantity of enzyme that produces one nmol of NO per min at 37°C in a solution containing 1 mM L-arginine, 1 mM Magnesium acetate, 5 µM oxyhemoglobin, 0.1 mM NADPH and 12 µM tetrahydro-L-biopterin.

Product Number: NS 10

cDNA Probe for Murine iNOS: This 3.9 kb fragment of the cDNA encoding murine inducible nitric oxide synthase is useful for the preparation, by nick translation or random priming, of labeled probes for Northern blot analyses. ( Science, 256:225-228, 1992)

Product Number: NS 02

Endothelial Cell Nitric Oxide Synthase

This NOS isoform is constitutivley expressed in endothelial cells (eNOS) and undergoes several post-translational modifications, including myristolation and palmitoylation, which cause the enzyme to become membrane-associated.

Anti-Endothelial Cell Nitric Oxide Synthase: This polyclonal antibody, developed against a peptide corresponding to residues 599 - 613 of the 140 kDa bovine eNOS, specifically reacts with this NOS isoform in human, rat, and mouse vascular endothelial cells. Useful for immunoprecipitation and Western blotting applications.

Product Number: NS 15

EZ-Prime Human Endothelial Nitric Oxide Synthase Primers: This oligonuceotide primer set is designed for specific PCR amplification of human eNOS message, and gives an amplimer of 485 base pairs. Each vial is sufficient for > 50 amplification reactions, and provides instructions and optimal reaction conditions.

Product Number: EZ 09

Neuronal Nitric Oxide Synthase

Neuronal NOS (nNOS, a.k.a. brain NOS or bNOS) is a 155 kDa cytosolic protein that is constitutively expressed, primarily in neuronal cells. NO synthesized by nNOS functions primarily as a neurotransmitter.

Neuronal Nitric Oxide Synthase, Recombinant Rat Brain: Recombinant nNOS was expressed in insect cells using a baculoviral vector in the presence of heme. The purified protein has a specific activity of approximately 0.5 µmol/mg/min at 37°C.

Product Number: NS 11

Anti-Neuronal Nitric Oxide Synthase: This polyclonal antibody, developed against a peptide corresponding to residues 724-739 of rat bNOS, is specific for the 155 kDa neuronal NOS in rat, mouse, and bovine cells. It is useful for immunoprecipitation and Western blotting applications.

Product Number: NS 12

EZ-Prime Human Neuronal Nitric Oxide Synthase Primers: This oligonucleotide primer set is designed for specific PCR amplification of human neuronal NOS message, and gives an amplimer of 471 base pairs. Each vial is sufficient for > 50 amplification reactions and provides instructions and optimal reaction conditions.

Product Number: EZ 08