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Setting up a kinase profiler with IMAP

Step 1. Add 5 L of the Inhibitor prepared in Step 2 to the + Inhibitor wells.

Step 2. Add 5 L of diluent prepared in Step 2 to the - Inhibitor wells.

Step 3. Add 5 L of enzyme dilutions prepared in Step 3 to the appropriate wells.

Step 4. Incubate as needed at room temperature to allow for Inhibitor and enzyme interaction.


Add any special components required by certain kinases (See Table A); for all other kinases, add Complete Reaction Buffer
As shown in Table A, add 5 L of a 4X stock of any special reaction requirements. Prepare all of these stocks in Complete Reaction Buffer.

Step 1. For AMPK: add 5 L of a 4X stock of 200 M AMP (50 M final concentration).

Step 2. For tyrosine kinases: add 5 L of 4X stock of 8 mM MnCl2 (2 M final concentration).


Mix together ATP and substrate in a 1:1 ratio and add to the assay plate to start the reaction.
Step 1. For the 100 M ATP wells shown in Table B, add 150 L of the 8X ATP solution prepared in Step 4 per 150 L of the 8X Substrate Solution prepared in Step 5 to make 300 L of a 4X ATP/Substrate Solution for each substrate. Add 5 L 4X ATP/Substrate Solution to the appropriate wells.

Step 2. For the 10 M ATP wells shown in Table B, add 150 L of the 8X ATP solution prepared in Step 4 per 150 L of the 8X Substrate Solution prepared in Step 5 to make 300 L of a 4X ATP/Substrate Solution for each substrate. Add 5 L 4X ATP/Substrate Solution to the appropriate wells.

Step 3. For the No ATP controls shown in Table B, a
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