PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Enhanced Selectivity*

Protocol summary

1. Prepare PPi Sensor working solution  (50 µL)
2. Add Pyrophosphate standards and/or test samples (50 µL)
3. Incubate at room temperature for 10 to 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 370/470 nm (Cutoff = 455 nm)

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

1. PPi Sensor stock solution (200X):
Add 50 µL of DMSO (Component D) into the vial of PPi Sensor (Component B) to make 200X PPi Sensor stock solution. Note: 25 µL of the PPi Sensor stock solution is enough for one 96-well plate.

 Add 25 µL of 200X PPi Sensor stock solution to 5 mL of Assay Buffer (Component A), and mix them well. Note: Due to the high sensitivity of this assay to PPi, it is important to use PPi-free labware and reagents. Note: DTT (1 uM) will increase the background, MgCl2 ≥ 2mM decrease the response.

Table 1. Layout of pyrophosphate standards and test samples in a solid black 96-well microplate.  PS = Pyrophosphate Standard, BL = Blank Control, TS = Test Sample. 

Table 2. Reagent composition for each well

1. Prepare Pyrophosphate standards (PS), 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 PPi Sensor working solution to each well of Pyrophosphate 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  PPi Sensor working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 370/470 nm (Cutoff = 455 nm)