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Setting up a kinase profiler with IMAP

assay, the lipid activation step will be omitted to streamline the protocol.

For Ros, the next kinase on our list, the substrate Rostide has already been validated in IMAP, so an SF Tyr assay was not necessary. The TIE2 assay was the last on the Table 1 profiler list not yet optimized for IMAP. TIE2 was assayed on the SF Tyr plate at 0.7 U/mL, and the results are presented in Figure 17, Panel A. There are more than a dozen substrates which can be phosphorylated by TIE2 at that concentration, as indicated by a DmP signal greater than 150 mP, so from these results it is difficult to choose the substrate that provides the most sensitive TIE2 IMAP assay. Testing with a lower concentration of TIE2 increased the stringency of the SF Tyr assay. The SF Tyr results with a reduced concentration (0.1 U/mL) of TIE2 are shown in Figure 17B.

Table 3. Top-Ranking TIE2 Substrates SF Tyr Well ID Peptide Sequence MDC Part Number (Name) SF Tyr 0.1 U/mL TIE2 (Figure 18B) Using SF Binding Solution TIE2 Titration (Figure 19) Using Optimized Binding Solution Bkgnd FP '"/>

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