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No reagent preparation is needed.
Plasma samples may be deproteinized before performing the assay.
Standard Preparation: Prepare the calibrate in 1 mL tubes following the Table 1 ( see in booklet) . Use ultrapure water as diluent. Performing the assay: 1. Add 50 μL of samples or standards in each well (96-well plate). 2.Add 50 μL of Reagent A in each well. Incubate for 10 minutes protected from light. 3.Add 50 μL of Reagent B in each well. Incubate for 10 minutes protected from light. 4.Read the absorbance at 540 nm within 30 minutes.
Determine average absorbance value of each experimental sample. Determine its concentration by comparison to the Nitrite Standard reference curve (Figure 2 see images). Nitrite (μM) = (?A540nm- intercept)/ slope