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Setting up a kinase profiler with IMAP

to test a single candidate inhibitor or activator compound with different concentrations of ATP, titrate the drug candidate with each enzyme, titrate each enzyme with a single concentration of the compound, test multiple compounds with a single ATP concentration and a single kinase concentration, or other protocols may be devised.

Profiling experiments may vary depending on the type of compounds being tested. For example, if the drug candidates to be profiled are known to be ATP-competitive inhibitors, it may be useful to test with two or more concentrations of ATP. To facilitate profiling experiments, kinases with similar KmATPs can be tested together. For determination of the apparent KmATP for each kinase, please refer to IMAP Application Note #3, Determination of Ki and Km values in IMAP Assays, at www.moleculardevices.com.

For our IMAP Profiler, we used the EC50 concentration of each kinase with both 10 M and 100 M ATP. Three known kinase inhibitors (H89, SB203580 and staurosporine) were tested with this IMAP Profiler. The plate set-up is shown in Table 5. IMAP Reaction Buffer containing 0.01% Tween 20 rather than 0.1% BSA as a carrier was utilized in this Profiler to avoid inhibitor binding to BSA. This type of binding could reduce the effective inhibitor concentration and result in a right-shifted IC50 curve. Although assays listed in Table 4 that utilized the 100% Buffer A/1:400 BR Binding Solution equilibrated within one hour, those assays with Binding Solutions containing greater than 25% Buffer B required more time to achieve a stable
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