Mps1-IN-2

Kinase experiment:

The kinase binding assay is used to assess compound binding to TTK by monitoring displacement of a fluorescently labeled, ATP site-directed kinase inhibitor (Kinase Tracer 236) from the kinase active site. Each 15 μL assay contains 5 nM TTK, variable amounts of test compound (Mps1-IN-2), 30 nM Kinase Tracer 236, 2 nM Eu-anti-GST Antibody, and 1% DMSO (residual from compound dilution) in Kinase Buffer A (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35). Binding assays are initiated by addition of 5 μL of test compound (from 2-fold dilution series) to 5 μL of a kinase/antibody mixture, followed by addition of 5 μL of antibody. Assay plates are read using using standard Eu-based TR-FRET settings with excitation at 340 nm and emission monitored at 615 nm (donor) and 665 nm (acceptor). Emission intensities are measured over a 200 µs window following a 100 µs post-excitation delay[1].

References:

[1]. Kwiatkowski N, et al. Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function. Nat Chem Biol. 2010 May;6(5):359-68.