Amplite® Colorimetric Ammonia Quantitation Kit *Blue Color*

Protocol summary

1. Prepare Ammonium Chloride standards or test samples (50 µL)
2. Add Assay Buffer I (50 µL)
3. Incubate at RT or 37°C for 5 min
4. Add Assay Buffer II (50 µL)
5. Incubate at RT for 30 - 60 min
6. Monitor Absorbance increase at 660 - 670 nm

Important notes
Thaw all the kit components to room temperature before starting the experiment.

Table 1. Layout of Ammonium Chloride standards and test samples in a clear bottom 96-well microplate. AS= Ammonium Chloride Standards (AS1 - AS7, 1 to 1000 µM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare Ammonium Chloride standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of Assay Buffer I (Component A) to each well of Ammonium Chloride standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Assay Buffer I into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature or 37°C for 5 minutes.
   
   
4. Add 50 µL of Assay Buffer II (Component B) to each well to make the total assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Assay Buffer II into each well instead, for a total volume of 75 µL/well.
   
   
5. Incubate the reaction at room temperature for 30 - 60 minutes.
   
   
6. Monitor the absorbance increase with an absorbance microplate reader at 660 - 670 nm. Note: The color turns to yellow after Assay Buffer II (Component B) is added, and the wells with Ammonium Chloride standard or samples will show bluish green color after incubation. The intensity of the color will reach the maximum in 30 - 60 minutes.