Receptor Tyrosine Kinase Inhibitor Profiling Using Bead-Based Multiplex Sandwich Immunoassays

Abstract

Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks. Here, we describe the analysis of the phosphorylation state of receptor tyrosine kinases using the epidermal growth factor receptor (EGFR) as an example. We provide a protocol for a bead-based sandwich immunoassay that enables a relative quantification of the EGFR and its generic tyrosine phosphorylation. We also present data from a kinase inhibitor experiment using 96-well cell-culture plates and a commercially available kit for the analysis of seven receptor tyrosine kinases.