Basic NeuroscienceShort communicationUtilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells

- The method utilizes sandwich-ELISA principles and advantages in an in situ context.
- The method specifically detects Trk receptor phosphorylation in cultured cells.
- In situ phospho-Trk ELISA can be utilized for research and compound screening.

BackgroundTrk 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 methodWe 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.ResultsIn 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 methodsIn situ phospho-Trk ELISA utilizes the principles and advantages of conventional sandwich ELISA in an in situ context.ConclusionsWe 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.