PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*

Protocol summary

1. Run kinase reaction (20 µL)
2. Add ADP Sensor Buffer (20 µL)
3. Add ADP Sensor (10 µL)
4. Incubate at room temperature for 15 minutes - 1 hour
5. Monitor fluorescence intensity

Important notes
Thaw all the six components at room temperature before use. Avoid direct exposure of ADP Sensor I (Component B1) to light. 

1. ADP Sensor I stock solution (50X):
Add 20 µL DMSO (Component B3) into vial of ADP Sensor I (Component B1).

2. ADP Sensor stock solution (1X):
Add 20 µL of 50X ADP Sensor I stock solution into vial of ADP Sensor II (Component B2).

3. ADP standard solution (300 mM):
Add 100 µL of H₂O into ADP Standard (Component C) to make a 300 mM ADP stock solution.

Table 1. Layout of ADP standards and test samples in a solid black 96-well microplate. SD = ADP standard (SD1 - SD7, 0.05 to 30 uM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

Run kinase reaction (Reagents are not provided for this step):

1. Prepare 20 µL of kinase reaction solution as desired. The components of kinase reaction should be optimized as needed (e.g., an optimized buffer system might be required for a specific kinase reaction). In most cases, ADP assay buffer (Component D) can also be used to run kinase reaction if you do not have the optimized kinase buffer.
   
   
2. The Amplite™ Fluorimetric ADP Assay Kit is used to determine the ADP formation. The ADP Sensor is unstable in the presence of thiols such as DTT and β-mercaptoethanol. Final thiol concentration higher than 10 µM would significantly decrease the assay dynamic range.
   
   

Run Amplite™ ADP assay:

1. Add 20 µL of ADP Sensor Buffer (Component A) and 10 µL of ADP Sensor stock solution into each well filled with the 20 µL kinase reaction solution to make the total ADP assay volume of 50 µL/well. Note: The ADP Sensor is unstable in the presence of thiols such as DTT and β-mercaptoethanol. Final thiol concentration higher than 10 µM would significantly decrease the assay dynamic range. Note: The ADP assay should be run at pH from 6.5 to 7.4.
   
   
2. Incubate the reaction mixture at room temperature for 15 minutes to 1 hour.
   
   
3. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm.