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 EC50
s 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
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
of substrate hits on the plate and facilitated in
choosing the profiling substrate.
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Table 2. Top-Ranking FAK Substrates
. Setting up the laboratory to avoid contamination2
. Reduce Errors and Save Time When Setting Up PCRs3
. Setting up Successful siRNA Library Screens4
. LiquiChip Kits for bead-based cytokine and kinase assays5
. Homogeneous, chemiluminescent kinase assay kits for assessing the functional activity of a broad range of serine-threonine and tyrosine kinases6
. TKXtra DataFile: Homogenous tyrosine kinase detection using High Efficiency Fluorescence Polarization