Amplite® Colorimetric Urea Quantitation Kit *Blue Color*

Protocol summary

1. Prepare urea standards or test samples (50 µL)
2. Add urea working solution (50 µL)
3. Incubate at room temperature or 37°C for 30 - 60 min
4. Add Assay Buffer II
5. Read Absorbance at 665 nm

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

1. Assay Enzyme Mix stock solution (100X):
Add 100 µL of ddH₂O into the vial of Assay Enzyme Mix (Component A) to make 100X Assay Enzyme Mix stock solution.

Add 50 μL of reconstituted Assay Enzyme Mix stock solution (100X) into 5 mL Assay Buffer I (Component B) to make urea working solution. Note: The urea working solution should be used promptly and kept from light. The assay sensitivity will be decreased with longer storage time. Fresh urea working solution is recommended for the best result.

Table 1. Layout of urea standards and test samples in a clear bottom 96-well microplate. US= Urea Standards (US1 - US7, 1 to 1000 µM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare urea standards (US), 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 urea working solution to each well of urea standard, blank control, and test samples to make the total urea assay volume of 100 µL/well. For a 384-well plate, add 25 µL of urea working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction for 30 - 60 minutes at room temperature or 37°C, protected from light.
   
   
4. Add 50 µL of Assay Buffer II (Component C) to each well so that the total assay volume is 150 µL/well. For a 384-well plate, add 25 µL Assay Buffer II (Component C) to each well, for a total assay volume of 75 µL/well.
   
   
5. Incubate at room temperature for 10 - 15 minutes, and monitor the absorbance increase at 660 - 670 nm using an absorbance microplate reader. Note: The color turns to yellow after Assay Buffer II (Component C) is added, and the wells with urea standard or samples will show blue-green color after incubation. The intensity of the color will reach the maximum in 15 - 30 minutes, and is proportional to the concentration of urea. Note: The final color is stable for ~1 hour in room temperature and the color intensity will decrease with time.