Imap screening express kit

Cellular kinase activities were evaluated using nonradioisotopic methods such as the off‐chip mobility shift assay or IMAP (Carna Biosciences, Kobe, Japan).11 A kinase inhibition profiling panel was produced on the basis of the kinase inhibition rates. In off‐chip mobility shift assay, compound solution was prepared in assay buffer (20 mmol/L HEPES, 0.01% Triton X‐100, 2 mmol/L DTT, pH 7.5) and incubated in a 384‐well plate at room temperature. The reaction was stopped by adding Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences, Kobe, Japan). The substrate peptide and the phosphorylated peptide in the reaction solution were separated and quantified by LabChip system (Perkin Elmer, MA, USA). In IMAP assay, compound solution was prepared in assay buffer (20 mmol/L HEPES, 0.01% Tween‐20, 2 mmol/L DTT, pH 7.4) and incubated in a 384‐well black plate at room temperature. IMAP binding reagent (IMAP Screening Express kit; Molecular Devices, CA, USA) was added and incubated for 30 minutes. The kinase reaction was evaluated at the fluorescence polarization.

Substrate/ATP/metal (4×) and kinase (2×) solutions were prepared using with assay buffer (20 mM HEPES, 0.01% Tween-20, 2 mM DTT, pH 7.4), and then mixed in 384-well black plates for 1 hour at room temperature. IMAP binding solution (IMAP Screening Express kit; Molecular Devices, Sunnyvale, CA, USA) was added to each well and incubated for 30 min. The level of kinase activity was then evaluated by fluorescence polarization at 485/530 nm (Ex/Em).