Amplite® Colorimetric Alkaline Phosphatase Assay Kit *Yellow Color*

Protocol summary

1. Prepare alkaline phosphatase standards and/or test samples (50 µL)
2. Add ALP working solution (50 µL)
3. Incubate at RT or 37°C for 10 - 30 minutes
4. Monitor absorbance increase at 400 nm

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

1. pNPP stock solution (100X):
Add 300 µL of distilled H₂O into the vial of pNPP (Component A). Mix well. The pNPP stock solution should be used promptly. Note: The solution should be good for 3 - 4 weeks if stored properly.

2. Alkaline Phosphate standard solution:
Add 100 µL of distilled H₂O with 0.1% BSA (H₂O - 0.1% BSA) to Alkaline Phosphatase Standard (Component C, 10 units) to generate a 100 units/mL Alkaline Phosphatase standard solution.

Add 50 μL of pNPP Stock solution (100X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.05 mL of pNPP working solution.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Table 1. Layout of Alkaline phosphatase standards and samples in a white/clear bottom 96-well microplate. AS = Alkaline Phosphatase Standards (AS1 - AS7, 0.1 to 100 mU/mL); BL=Blank Control; TS=Test Samples

Table 2. Reagent composition for each well.

In supernatants:

1. Prepare alkaline phosphate 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 pNPP working solution to each well of alkaline phosphate standard, blank control, and test samples to make the total alkaline phosphate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of pNPP working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at the desired temperature for 10 to 30 minutes, protected from light.
   
   
4. Monitor the absorbance increase with an absorbance plate reader at 400 nm.
   
   

In cells:

1. Treat the cells as desired.

2. Add equal volume of pNPP working solution into each cell well (such as 100 µL/96-well plate or 50 µL/384-well plate). Note: Alternatively, remove the growth medium from the cell plate, and make 1:1 dilution of the 5 mL working solution with 5 mL distilled H₂O. Then add 100 µL (for a 96-well plate) or 50 uL (for a 384-well plate) of 1:1 diluted working solution to the cell wells.

3. Incubate the reaction at the desired temperature for 30 to 60 minutes, protected from light.

4. Monitor the absorbance increase with an absorbance plate reader at 400 nm.