BDP5290

MRCKα, MRCKβ, ROCK1 and ROCK2 assays are performed using an IMAP fluorescence polarization assay format. 8 to 12 nM of each kinase is incubated for 60 min at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide in the presence of 1 μM ATP and 0.5 mM MgCl₂ in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT (MRCKα and β); or 1 μM ATP, 10 mM MgCl₂ in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA 0.01% Triton X-100 and 1 mM DTT (ROCK1 and ROCK2). Typically, dose response analysis are performed over concentration ranges from 0.005 to 100 μM. Reactions are stopped by adding 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1×IMAP binding buffer. After 30 min incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization is measured on a plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition is calculated using no inhibitor and no enzyme controls as 0 and 100% inhibition, respectively. Kinase selectivity profiling is performed by Eurofins with 10 μM ATP and 10 μM BDP5290^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

MDA MB 231 or SCC12 cells are plated in a 96 well plate and cultured for 24 hours. Cells are then cultured for 24 hours in SCC12 medium with DMSO vehicle or indicated concentrations of BDP5290 in an IncuCyte ZOOM. Pictures are taken every 3 hours and confluence is measured using the IncuCyte analysis software. AlamarBlue is added to the medium and the cells are cultured for an additional day. Absorbances at 570 nm and at 600 nm are measured to assess cell health^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.