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Setting up a kinase profiler with IMAP

F Ser/Thr 2 kit has a protocol and set of peptide substrates distinct from the SF Ser/Thr 1 and SF Tyr kits. These IRAK 4 results are shown in Figure 8, Panel B. The four peptides on the IRAK SF Ser/Thr 2 plate that provided the greatest DmP were ID C3D4, A9B10 and G9H10. A9 B10 is the SF Ser/Thr 2 ID for the same FAM-PKCe pseudosubstrate-derived peptide discussed above, which underscores the reproducibility among IMAP SF kits. There are nine substrates common to both the SF Ser/Thr 1 and SF Ser/Thr 2 kits because they were described as substrates for kinases from both areas of the kinome. (See Figure 1.) Figure 9 shows the corresponding IRAK 4 dilution curves with the SF Ser/Thr 1 and SF Ser/Thr 2 substrates. The FAM-PKCe-derived peptide, of sequence 5FAM-ERMRPRKRQGSVRRRVNH2, had the lowest EC50 of all peptides tested. This peptide was therefore chosen to be the IRAK 4 profiling substrate.

JNKa2 is another kinase that does not yet have available substrates optimized for IMAP. For initial assay development, usable substrates for JNK2a2 were determined with the SF Ser/Thr 2 kit. (See Figure 10.)

As only 0.17 U/mL JNK2a2 was assayed on the SF Ser/Thr 2 plate, few peptides were hits in this assay. The EGFR-derived peptide (well ID E17-F18), an IMAP-validated substrate already in MDC's inventory, provided the sole signal greater than 100 mP. To confirm that this peptide would i
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