Nitric Oxide Quantitation Kit
faster, more accurate measurement of nitric oxide production
Active Motif’s Nitric Oxide Quantitation Kit is faster and easier-to-use than existing methods for measuring the production of nitric oxide in biological samples. The kit employs an innovative cofactor technology that reduces the time and number of steps required. The assay is also linear over a broader range of sample concentrations, which means that you’ll have to dilute and re-assay your samples far less frequently than with other methods (see Data tab). The kit can be used with a variety of sample types, including plasma, serum, saliva, urine, cell lysate, tissue homogenate and tissue culture medium.
Nitric oxide (NO) is a biological signaling and effector molecule involved in many biological functions, including maintaining blood pressure, modulating neural transmissions and stimulating immune responses. Nitric oxide is also implicated in the regulation of hypoxia-inducible factor 1α, a transcription factor that is a key regulator of oxygen homeostasis1, 2 (see TransAM® HIF-1). Consequently, Nitric oxide has become the subject of extensive research.
Figure 1: Dynamic range of nitric oxide standard curves.
- Huang, LE et al. (1999) J. Biol. Chem. 274: 9038-9044.
- Liu, Y et al. (1998) J. Biol. Chem. 273: 15257-15262.
Principle behind traditional nitric oxide assays
Nitric oxide has an extremely short half-life (< 10 seconds), which makes it difficult to detect and study directly. However, because nitric oxide is metabolized to nitrite and nitrate, quantitation of these stable anions can be used to indirectly determine the amount of nitric oxide originally present.
Typically, nitric oxide assays are performed using either a two-step assay or a three-step lactate dehydrogenase (LDH) assay. In both methods, the first step is the reduction of nitrate into nitrite by nitrate reductase. In the final step, Griess Reagent converts the nitrite into a purple-colored azo compound, which is quantitated by spectrophotometer at A540 (Figure 1). However, excess NADPH, an essential cofactor in nitrate reduction, interferes with the Griess reaction, limiting the sensitivity of the two-step assay. To improve sensitivity, an LDH step that degrades NADPH can be added before the Griess Reagent step, resulting in the more time consuming, more complicated three-step LDH assay.
Figure 1: The principle of nitric oxide assays.
Unique features of the Nitric Oxide Quantitation Kit
Active Motif’s Nitric Oxide Quantitation Kit offers advantages in time, ease-of-use and accuracy over the methods described above. It employs an innovative cofactor technology that accelerates the conversion of nitrate to nitrite while simultaneously degrading NADPH. Thus, the reductase reaction is reduced to only 30 minutes and sensitive colorimetric quantification can be performed without the need for LDH treatment. Moreover, the Active Motif method is linear over a broader dynamic range, which increases the range of sample concentrations that can be measured accurately (Figure 1). This means that you’ll need to dilute and re-assay fewer samples. For additional information, please download the Nitric Oxide Quantitation Kit manual.
Advantages of the Nitric Oxide Quantitation Kit
- Faster – Cofactor technology accelerates the nitrate reductase reaction
- Simpler – Provides high sensitivity in fewer steps
- More accurate – Linear results over a wider range of sample concentrations
- Scalable – 96-well format enables high-throughput automation, if desired
Contents & Storage
The Nitric Oxide Quantitation Kit contains Nitrate Reductase, Cofactors, Griess reagents and two 96-well plates, which are stored and guaranteed for 6 months at 4°C. Upon reconstitution, the Nitrate Reductase and Cofactors are stored and guaranteed for 2 months at -20°C.