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Setting up a kinase profiler with IMAP

ndeed be the profiling substrate chosen, JNK2a2 was titrated with the EGFR-derived peptide and the second- and third-ranked peptides, well IDs C3-D4 and A5-B6, respectively. A5B6, known as CDK7tide, is already in the IMAP inventory. Figure 11 shows that the EGFR-derived peptide performed better than the other two substrates tested, confirming the SF Ser/Thr 2 assay results in Figure 10. This peptide was chosen for the JNK2a2 profiling assay.

The next enzyme on the profiler list in Table 1 is the kinase NEK2. NEK2 can phosphorylate PLM-derived peptide, of sequence 5FAMIRRLSTRRR- COOH, but it would be costefficient to develop a more sensitive assay if possible. To this end, we assayed NEK2 on the SF Ser/Thr 2 plate. (See Figure 12.)

From the histogram shown in Figure 12, peptide with ID G9-H10 had a DmP of 269 mP, the highest substrate signal in the assay. This peptide, of sequence 5FAM-GTFRSSIRRLSTRRRCOOH, has more phosphorylation sites and a sequence extended from the FAM-PLM-derived peptide. The latter peptides DmP in the SF Ser/ Thr 2 assay was only 51 mP (labeled ID G9H10 on the SF Ser/Thr 2 plate). As is evident from
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