PhosphoWorks™ Fluorimetric ATP Assay Kit

Protocol summary

1. Prepare ATP working solution (50 µL)
2. Add ATP standards or test samples (50 µL)
3. Incubate at room temperature for 10 - 30 minutes
4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

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

1. Amplite^TM Red Substrate stock solution (200X):
Add 30 µL of DMSO (Component E) into the vial of Amplite^TM Red Substrate (Component A) to make 200X Amplite^TM Red Substrate  stock solution.

2. ATP standard solution (10 mM):
Add 0.5 mL of ddH2O into the vial of ATP Standard (Component D) to make 10 mM ATP standard solution.

1. Add 5 mL of Assay Buffer (Component C) into Enzyme Mix bottle (Component B), and mix well.

2. Add 25 µL of 200X Amplite^TM Red Substrate stock solution to the Enzyme Mix bottle, mix well to make APT working solution.

Table 1. Layout of ATP standards and test samples in a solid black 96-well microplate. AS= ATP Standards (AS1 - AS7, 0.14 to 100 µM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well. 

1. Prepare ATP 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 of ATP working solution to each well of ATP standard, blank control, and test samples to make the total ATP assay volume of 100 µL/well. For a 384-well plate, add 25 µL of ATP 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=540/590 nm (Cutoff = 570 nm).