A proximity ligation assay using transiently transfected, epitope-tagged proteins: application for in situ detection of dimerized receptor tyrosine kinases

摘要: The development of small molecule and antibody inhibitors targeting the interaction receptor tyrosine kinases (RTKs), such as epidermal growth factor (EGFR), is high pharmacological biological interest. Unfortunately, conventional biochemical techniques using cell or tissue lysates co-immunoprecipitation experiments to investigate EGFR dimerization are not always conclusive. Here we describe a series technical validation demonstrating utility proximity ligation assay (PLA)-based methodology for in situ visualization quantification ligand-dependent intact cells. Using PLA approach combined with universally applicable epitope tagging strategy, detected dimers cells transiently co-expressing FLAG-tagged MYC-tagged human EGFRs. Our data strongly suggest that can be used detect this signal generated protein interaction-based manner, rather than solely due target proteins. This application represents generalized RTK expression strategy protein-interaction analysis transient system where epitopes known unique enough discriminate between partners. also holds promise general screening tool specimens identify potential clinical relevance.