SunRed™ Phosphate

Protocol summary

1. Prepare and add 10 - 50 µM SunRed™ Phosphate in Tris buffer (50 µL)
2. Add alkaline phosphatase standards and/or test samples (50 µL)
3. Incubate at room temperature or 37°C for 30 to 120 minutes
4. Monitor fluorescence intensity at Ex/Em = 620/660 nm (cut off 640 nm)

Important notes
The following is the recommended protocol for alkaline phosphatase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.

1. SunRed™ Phosphate stock solution:
Prepare a 2 to 10 mM stock solution of SunRed™ Phosphate in sterile water. Note: The stock solution should be used promptly. Note: Do not use DMSO, ETOH or METH to make stock solution since it significantly increases assay background. Note: Protect from light.

SunRed™ Phosphate working solution (2X):
On the day of the experiment, either dissolve SunRed™ Phosphate solid in sterile H₂O or thaw an aliquot of the SunRed™ Phosphate 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. SunRed™ Phosphate final concentration of 5 to 25 μM is recommended for measuring alkaline phosphatase activity in solution.

1. Add 50 µL of 2X SunRed™ Phosphate working solution into each well of the alkaline phosphatase standard, blank control, and test samples to make the total alkaline phosphatase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of sample and 25 µL of 2X SunRed™ 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 Ex/Em = 620/660 nm (cut off at 640 nm) with a fluorescence plate reader.