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Setting up a kinase profiler with IMAP

ont-family:Symbol'>DmP and EC50 was the FAMSrc- Family-tide, so this peptide was chosen for the profiling assay.

As shown in Table 2, the FAM-Src-Family-tide on the SF Tyr plate had an FP background of > 200 mP with the 40% Buffer A, 60% Buffer B, 1:1500 BR Binding Solution as directed in the SF Tyr protocol. This FP background was optimized by testing the peptide only with different Binding Solutions (data not shown), as outlined in "IMAP Application Note #1: The IMAP Progressive Binding System." To achieve the FP background of 73 mP in the FAK titration assay, we used a Binding Solution of 10% Buffer A, 90% Buffer B and 1:1500 BR. This simple Binding Solution optimization reduced the background FP and maximized the FP signal. The Binding Solutions recommended by the SF Tyr kit protocol are usable but may not be optimal for each substrate; they were devised to allow for acceptable assay windows for all SF substrates while keeping the total number of SF Tyr assay Binding Solutions to a minimum.

The next kinase on the profiling panel without a known IMAP substrate is IRAK 4. As shown in Figure 8, Panel A, three substrates performed significantly better than the others in a SF Ser/ Thr 1 assay with this kinase. These peptides were: ID A11B12, C11D12 and K11L12 on the SF Ser/Thr 1 plate. K11L12 is the FAM-PKCe pseudosubstrate-derived peptide, of sequence 5FAM-ERMRPRKRQGSVRRRV-NH2, and is already available in the IMAP substrate inventory.

We also tested IRAK 4 with the SF Ser/Thr 2 kit for CK1, TKL and CMGC Ser/Thr Kinases because IRAK4 is a member of the TKL family. The S
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