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Setting up a kinase profiler with IMAP

dd 150 L of Complete Reaction Buffer per 150 L of the 8X Substrate Solution prepared in Step 5 to make 300 L of a 4X Substrate Solution for each substrate. Add 5 L 4X Substrate Solution to the appropriate wells.

Step 4. For the Buffer Only background controls, add 20 L of Complete Reaction Buffer to the appropriate wells. Each well of the assay should now have 20 L volume.


Plate Incubation
Step 1. Cover the plate and protect from light. Incubate at room temperature for 3060 minutes.

Note: You may need to optimize reaction time for your individual needs.


C: ADD BINDING SOLUTION AND READ FP
Progressive Binding Solutions preparation
Prepare the Progressive Binding Solutions according to the plate diagram in Table 5

Note: For Binding Solution optimization information, please refer to the IMAP Progressive Binding System Application Note. Binding Solutions are listed as % Progressive Buffer A/% Progressive Buffer B/dilution of Progressive BR.

Step 1. Prepare 15 mL of 100/0/1:400 Binding Solution by adding 37.5 mL BR to 15 mL of 1X Buffer A.

Step 2. Prepare 15 mL of 75/25/1:600 Binding Solution by adding 25 mL BR to 11.25 mL of 1X Buffer A + 3.75 mL of 1X Buffer B.

Step 3. Prepare 10 mL of 60/40/1:1200 Binding Solution.

a. Mix 6 mL of 1X Buffer A with 4 mL 1X Buffer B to make Binding Buffer.

b. Add 30 L of BR to 270 L of 0.1 N HCl and mix. This is an intermediate 1:10 dilution of the BR into 0.1 N HCl. Save the in
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