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Setting up a kinase profiler with IMAP

F Ser/Thr 1 plate, as shown in Figure 4. Because only one kinase concentration is tested in the Substrate Finder, the SF Ser/Thr 1 results do not completely reflect the efficiency of each substrate; the EC50s must be determined. Therefore, we titrated Aurora B with PKAtide and peptide G3-H4. These results are shown in Figure 5. The performance of the two substrates is very comparable, so we chose the G3-H4 peptide as our Aurora B profiling substrate. Aurora B is the substrate of choice for AMPK, another of our profiling enzymes, thereby reducing the number of different substrates required for the profiling screen.

It is worth noting that, overall, the AMPK SF Ser/Thr 1 assay background and calibrator control FPs (Figure 2) were reproducible in the Aurora B SF Ser/Thr 1 assay (Figure 4). This highlights the ease of assay development with IMAP.

IMAP assays for the kinases CDK5/p25 and Casein Kinase 1 (CK1) have already been developed with MDC inventory substrates, as shown in Table 1. In contrast, a validated IMAP substrate for FAK had not yet been found, so the IMAP SF Tyr plate was used to find one. The SF Tyr assay has a distinct protocol and set of peptide substrates that are different from those of the SF Ser/Thr 1 and SF Ser/Thr 2 kits. As shown in Figure 6, the initial assay with 0.07 U/mL FAK (Panel A) was then repeated with 0.02 U/mL (Panel B). Assaying with the lower concentration of enzyme reduced the number of substrate hits on the plate and facilitated in choosing the profiling substrate.


Table 2. Top-Ranking FAK Substrates
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