Amplite® Universal Fluorimetric Kinase Assay Kit *Red Fluorescence*

Protocol summary

1. Run kinase reaction (20 µL)
2. Add ADP Sensor Buffer (20 µL)
3. Add ADP Sensor working solution (10 µL)
4. Incubate at room temperature for 15 minutes - 1 hour
5. Monitor fluorescence Intensity at Ex/Em = 540/590 nm (Cutoff = 570nm)

Important notes
Thaw all the six components at room temperature before use. Black plates are strongly recommended to achieve the best results. The ADP assay should be run at pH from 6.5 to 7.4. Avoid direct exposure of ADP Sensor I (Component B1) to light. Avoid potential ADP contamination from exogenous biological sources.

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

2. ADP standard solution (300 mM):
Add 100 µL of ddH₂O into ADP Standard (Component C) to make a 300 mM ADP standard solution. 

Add 50 µL of 50X ADP Sensor I stock solution into vial of ADP Sensor II (Component B2) to make ADP Sensor working solution. Note: The reconstituted ADP sensor is not stable, make fresh as needed.

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

1. Prepare 20 µL (or 10 µL for 384-well plate) of kinase reaction solution/well 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 Kinase Assay Kit is used to determine the ADP formation.

Run Amplite™ ADP assay:

1. Add 20 µL of ADP Sensor Buffer (Component A) and 10 µL of ADP Sensor working solution into each well filled with the 20 µL kinase reaction solution to make the total ADP assay volume of 50 µL/well. For a 384-well plate, add 10 µL of ADP Sensor Buffer (Component A) and 5 µL of ADP Sensor into each well filled with the 10 µL kinase reaction solution to make the total ADP assay volume of 25 µL/well.
   
   
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  (Cutoff = 570 nm).

Generate an ADP calibration curve (Not required for the screening of kinase inhibitors):

1. Add 20 µL (for a 96-well plate) or 10 µL (for a 384-well plate)/well of ADP Sensor Buffer (Component A) and 10 µL (for a 96-well plate) or 5 µL (for a 384-well plate) of ADP Sensor into each well of serially diluted ADP standards to make the total volume of 50 µL (for a 96-well plate) or 25 µL (for a 384-well plate) for each reaction.
   
   
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 (Cutoff = 570 nm).
   
   
4. Generate an ADP standard curve.