RTK Phosphorylation

Abstract

A homogeneous multiplex immunoassay for RTK phosphorylation state that is adaptable to HTSJane Schmidt, Ph.D., Judy Haning, Shaya Anderson, Richard Fuerstenberg. R&D Systems, Inc. Minneapolis, MNReceptor Tyrosine Kinases (RTKs) are widely expressed transmembrane protein receptors for various extracellular signaling molecules. Binding of a specific ligand leads to phosphorylation at specific sites in the cytoplasmic tail which triggers further signal transduction processes. RTKs play critical roles in the regulation of developmental processes including cell survival, proliferation, and motility, which are involved in cancer formation when not properly regulated. For these reasons, RTKs have become popular therapeutic targets for drug development. The Human RTK VersaMAP Magnetic Multiplex System kits are bead‐based multiplex immunoassays for the analysis of RTK phosphorylation. Due to the homogeneous assay format and small sample volume, these assays can be readily adapted for high‐throughput screening with minimal manual intervention from cell treatment to data collection. To demonstrate this, several different cell lines were grown in 96‐well plates, treated with various stimulants or inhibitors, and lysed in the same plate. Diluted lysates were transferred to the assay plate for analysis of RTK phosphorylation by Human RTK VersaMAP assays in two multiplexes of 25 each. Using these assays, relative phosphorylation states were successfully determined for the various cells and treatments.