NOSdetect Assay Kit - Details & Specifications

The NOSdetect Assay Kit is a simple, sensitive and specific assay for nitric oxide synthase (NOS) activity. The NOSdetect assay kit is based on the biochemical conversion of L-arginine to L-citrulline by NOS. This reaction, which represents a novel enzymatic process, involves a five-electron oxidation of a guanidinonitrogen of L-arginine to nitric oxide (NO), together with the stoichiometric production of L citrulline (Figure 1). The reaction consumes 1.5 equivalents of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and also requires molecular oxygen, calcium, calmodulin and tetrahydrobiopterin (BH4).

Measuring NOS activity by monitoring the conversion of arginine to citrulline is currently the standard assay for NOS activity in both crude and purified enzyme preparations. Advantages of the NOSdetect assay kit include the use of radioactive substrates ([3H]arginine or [14C]arginine) that enable sensitivity to the picomole level, as well as the specificity of the assay for the NOS pathway due to the direct enzymatic conversion of arginine to citrulline in eukaryotic cells. Additionally, the easy separation of neutrally charged citrulline from positively charged arginine allows multiple assays to be performed easily.

For routine assays, radioactive arginine is added to intact tissues or protein extracts. After incubation, the reactions are stopped with a buffer containing ethylenediaminetetraacetic acid (EDTA), which chelates the calcium required by NOS and, consequently, inactivates the NOS. Equilibrated resin, which binds to the arginine, is added to the sample reactions and the reactions are then pipetted into spin cups. The citrulline, being ionically neutral at pH 5.5, flows through the cups completely. The NOS activity is then quantitated by counting the radioactivity in the eluate.

For Research Use Only. Not for use in diagnostic procedures