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Setting up a kinase profiler with IMAP

uffer A/1:400 BR Binding Solution equilibrate within one hour, those assays with Binding Solutions containing greater than 25% Buffer B require more time to achieve a stable signal. To simplify the protocol, the entire Profiler assay is incubated overnight with Binding Solution.


12-KINASE PROFILING SCREEN PROTOCOL
A: PREPARE SOLUTONS

Prepare Reaction Buffer
Step 1. All IMAP reagents are available in regular and bulk quantities. Please refer to the protocol included with each reagent for proper storage and preparation of the working stock. Add DTT to IMAP 0.01% Tween 20 Reaction Buffer for a final concentration of 1 mM. This is the Complete Reaction Buffer.


Prepare Inhibitor Working Solutions and the No Inhibitor Control Solution
Step 1. Dilute test compounds to 4X the final concentration in Complete Reaction Buffer. If a diluent other than Complete Reaction Buffer is used in the profiling assay, we recommend that each enzyme be first tested for its tolerance to this diluent. Some commonly used inhibitor diluents include DMSO and EtOH, which are both compatible with the IMAP system.

Step 2. For this assay, inhibitor stocks were at 110 mM in DMSO, and an amount of DMSO equivalent to the inhibitor stock volume was added to Complete Reaction Buffer for addition to the No Inhibitor control wells.


Prepare the Enzyme Working Stock Solutions
Step 1. Calculate concentration according to the enzyme-specific activity given in the suppliers Certificate of Analysis. For example: 2 g enzyme in 50
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