FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*

Protocol summary

1. Prepare 10 - 50 µM Phosphate in Tris buffer (50 µL)
2. Add phosphatase standards and/or test samples
3. Incubate at room temperature or 37°C for 30 to 120 minutes
4. Monitor fluorescence intensity at Ex/Em= 490/514 nm

Important notes
Phosphatase substrates can be detected by phosphatases that may not be specifically listed. The following is the recommended protocol for phosphatase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.

1. FDP stock solution:
Prepare a 2 to 10 mM stock solution in ddH₂O. Note: The stock solution should be used promptly.

FDP working solution (2X):
On the day of the experiment, either dissolve FDP in ddH₂O or thaw an aliquot of the stock solution at room temperature. Prepare a 2X working solution of 10 to 50 µM in 100 mM Tris buffer or buffer of your choice, pH 8 to 9 (not phosphate buffer).

1. Add 50 µL of 2X FDP working solution into each well of the phosphatase standard, blank control, and test samples to make the total phosphatase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of sample and 25 µL of 2X Phosphate working solution into each well.
   
   
2. Incubate the reaction for 30 to 120 minutes at the desired temperature, protected from light.
   
   
3. Monitor the fluorescence increase at an appropriate filter set with a fluorescence plate reader.
   
   
4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the phosphatase reactions.