Step 2. An EC50 concentration (predetermined by enzyme titration assay) of each kinase is suggested for the screen set up in Table B. Prepare a working stock of 150 mL of 4X the EC50 concentration for each kinase on one plate.
Prepare the ATP Working Solutions
Step 1. To make an 800 M ATP working solution, add 0.16 mL of a 10 M stock of ATP per 1.84 mL of Complete Reaction Buffer.
Step 2. To make an 80 M ATP working solution, add 0.1 mL of the 800 M solution prepared in Step 4A to 0.9 mL of Complete Reaction Buffer.
Step 3. The resulting dilutions of 800 and 80 M are 8X the final reaction concentrations of 100 and 10 M ATP.
Prepare the Substrate Working Solutions
For this assay, there are 10 different substrate solutions to prepare. Refer to Table A for details.
Step 1. Prepare each 20 M Fluorescent Substrate solution in Reaction Buffer with no DTS. Aliquot and store substrates at -20C to asssure stability for future use.
Step 2. To make an 800 nM substrate working solution, add 20 L of each 20 M solution per 480 L of Complete Reaction Buffer. Aliquot 3 x 150 L per microtube. This solution is 8X the final reaction concentration of 100 nM substrate.
B: SET UP KINASE REACTION
Add components to the 384-well assay plate according to the plate diagram in Table B