Cell treatment and lysis in 96-well filter-bottom plates for screening Bcr-Abl activity and inhibition in whole-cell extracts.

Although conventional high-throughput screens performed in vitro with purified protein kinases are powerful tools to discover new kinase inhibitors, they are far from ideal for determining efficacy in vivo. As a complementary approach, cell-based, target-driven secondary screens may help predict in vivo compound potency and specificity as well as evaluate bioavailability and toxicity. Here the authors report a simple protocol for treating K562 Bcr-Abl-expressing cells with small-molecule kinase inhibitors in 96-well filter-bottom plates followed by in-plate cell lysis. The lysates were assayed via a solid-phase kinase assay, allowing determination of apparent IC(50) for known Bcr-Abl inhibitors as well as facilitating the screening of a small kinase inhibitor library. This approach may have further applications in generating lysates for analyzing kinase activity and inhibition in other nonadherent suspension cell lines.