Patel, P.R., Sun, H., Li, S.Q., Shen, M., Khan, J., Thomas, C.J. and Davis, M.I.
Notes: This article describes how a miniaturized high-throughput biochemical assay for Yes1 kinase was developed and used for screening inhibitors in small molecule libraries. Kinase activity of Yes1 was assessed by dispensing 2µl of Yes1 enzyme in a 1,536-well white multiwell plate and mixing with compounds and substrate for a total kinase reaction volume of 25µl. The amount of ADP was quantitated using the ADP-Glo™ Kinase Assay and normalized to vehicle and minus-enzyme controls. Some of the inhibitors identified in the in vitro assay were tested in a cell-based assay. Two rhabdomyosarcoma cell lines, RD and RH30, were seeded at 4,000 cells/well in a 96-well plate with 100µl of culture medium. After an overnight incubation, 100µl of medium with 0, 0.1, 1, 5, 10, 15 or 20μM of inhibitor (final concentration) was added. After 48 hours, the number of cells was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (4354)