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Setting up a kinase profiler with IMAP

ng results reflect this also. SB203580 has been shown to inhibit JNK2 activity, albeit at a higher concentration than required for p38.4 Although JNK2a2 was almost completely inhibited by SB203580 in the presence of 10 M ATP, the inhibitor was less effective when tested in the profiling assay with 100 M ATP. Godl et al. reported that half-maximal JNK2 inhibition occurred at 11 M in an in vitro kinase assay with 100 M ATP, but dropped to 0.7 M when assayed with only 2 M ATP. 6 These results are consistent with Figure 21 profiling assay data.

We also tested the compound H89 in the IMAP profiling assay, as shown in Figure 22. At the 20 M assay concentration for H89, AMPK and Aurora B activities were significantly inhibited, as expected from previous studies.(7,8) According to Davies et al., AMPK was inhibited more than 75% by 10 M H89, which correlates with the 80% inhibition of AMPK at 10 M ATP observed in this assay.5 H89 is also known to inhibit CK1, according to Chijiwa et al., who calclulated the Ki to be 38.3 M.9 In our profiling assay, CK1 activity was significantly blocked by this compound.


CONCLUSIONS
This Application Note summarizes how to efficiently set up and run a profiling screen with IMAP. As we show here, the profiling results with the commercially available compounds staurosporine, SB203580 and H89 further demonstrate that IMAP provides inhibition data highly consistent with other in vitro kinase assay methods. Many IMAP substrates are now available as validated substrates, which enables a wide
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