PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*

1. Prepare Pyrophosphate standards and/or test samples (50 µL)
2. Add Pyrophosphate working solution (50 µL)
3. Incubate at room temperature for 10 to 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 316/456 nm (Cutoff = 420 nm)

Thaw all the four components at room temperature before use.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

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

Add 10 µL of 50 mM Pyrophosphate Standard (Component C) into 490 µL of ddH₂O or 50 mM Hepes buffer (pH 7) to make 1 mM Pyrophosphate standard solution.

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

Table 1. Layout of Pyrophosphate standards and test samples in a solid black 96-well microplate. PS = Pyrophosphate Standard (PS1 - PS7, 0.14 to 100 µM), 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 working solution to each well of Pyrophosphate standard, blank control, and test samples to make the total Pyrophosphate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of PPi working solution into each well instead, for a total volume of 50 µL/well. Mix the reagents thoroughly.
3. Incubate at room temperature for 10 to 30 minutes.
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 316/456 nm (Cutoff = 420 nm).