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Setting up a kinase profiler with IMAP

L with specific activity of 1000 units per mg provides 40 units/mL, where 1 unit is defined as 1 nmol phosphate incorporated into substrate per minute.

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

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