title: 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.
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany. poetz@nmi.de
authors.forename: Oliver
authors.initials: O
authors.lastname: Pötz
authors.forename: Nicole
authors.initials: N
authors.lastname: Schneiderhan-Marra
authors.forename: Tanja
authors.initials: T
authors.lastname: Henzler
authors.forename: Thomas
authors.initials: T
authors.lastname: Herget
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Joos
mesh_terms: D000906:Antibodies / Q000378:metabolism; D019921:Biotinylation; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D019073:HT29 Cells; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008863:Microspheres; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011933:Reagent Kits, Diagnostic; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000906:Antibodies; D047428:Protein Kinase Inhibitors; D011933:Reagent Kits, Diagnostic; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases