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Setting up a kinase profiler with IMAP

this note, lower enzyme concentrations were employed to increase the stringency of the assay and reduce the number of substrate hits.

The results in Figure 2 show that several of the tested substrates will provide an AMPK activity assay with a DmP of greater than 300 mP at 1 U/mL AMPK. These peptides were then compared with an enzyme titration to identify the most sensitive assay as measured by the EC50. (See Figure 3.) The lowest EC50 in the assay was 0.01 U/mL, obtained with the peptide 5FAM-LKKLRRRLSDANF-NH2 (ID G3H4). This peptide was chosen as the AMPK profiling substrate because its assay both conserved enzyme and maintained a robust signal. As AMPK is maximally active in the presence of AMP, AMP was added to the enzyme titration assay and will be used in the profiling assay at a concentration of 50 M.

The Aurora B assay on the Table 1 panel had also not yet been developed with IMAP. This kinase was tested with the SF Ser/Thr 1 kit to determine efficient substrates.

The Aurora B assays with peptides ID K7L8 (5FAM-GRTGRRNSI-NH2, PKAtide, IMAP validated) and ID G3H4 (5FAMLKKLRRRLSDANF- NH2) resulted in the greatest DmP values on the S
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