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Setting up a kinase profiler with IMAP

Figure 13, peptide G9H10 provides a > 20- fold more sensitive assay than the PLM-derived peptide, significantly reducing the cost-per-well of the NEK2 profile assay.

Another kinase planned for the profiling assay is PAK4. To find the best IMAP substrate for this enzyme, we assayed PAK4 with the SF Ser/Thr 2 kit. (See Figure 14.)

The SF Ser/Thr 2 assay with PAK4 had three peptides with a DmP result greater than 150 mP: well IDs C9D10, O9P10 and A13B14. Peptide C9D10 and O9P10 had very similar results in the SF Ser/Thr 2 assay, which were echoed in the PAK4 titration assay. The peptide with the slightly better EC50 was C9D10, so that substrate was chosen to be the PAK4 profiling peptide.

There is an inventory substrate available for PKCx, which is a PKC isoform that is further activated by lipids. This FAM-PKCe-derived peptide (5FAM-ERMRPRKRQGSVRRRV-NH2) is also the profiling substrate for IRAK4. Figure 16 shows that DAG and PS are not necessary for a robust PKCx assay. For the kinase profiling
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