Utilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells.

BACKGROUND Trk receptor tyrosine kinases regulate multiple important neuronal processes during the development and in the adulthood. Tyrosine phosphorylation of Trk serves as the initial step in the Trk signaling pathway and indicates receptor' autocatalytic activity. However, methods allowing simple and large-scale Trk phosphorylation analyses in cultured cells are lacking. NEW METHOD We describe an in situ phospho-Trk ELISA (enzyme-linked immunosorbent assay) method where cell culture, receptor stimulation and Trk phosphorylation analysis are all performed on the same multiwell plate. RESULTS In situ phospho-Trk ELISA readily and specifically detects neurotrophin-induced Trk phosphorylation in cultured cells. A proof-of-concept small molecule screening of a library composed of 2000 approved drugs and other bioactive compounds was carried out using this novel method. COMPARISON WITH EXISTING METHODS In situ phospho-Trk ELISA utilizes the principles and advantages of conventional sandwich ELISA in an in situ context. CONCLUSIONS We describe a novel method that can be efficiently used to examine Trk receptor phosphorylation in cultured cells. Principally similar methods can be developed to examine the levels and signaling of any intracellular protein.