Abstract
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.
| Original language | English |
|---|---|
| Journal | Journal of Neuroscience Methods |
| Volume | 222 |
| Pages (from-to) | 142-6 |
| Number of pages | 5 |
| ISSN | 0165-0270 |
| DOIs | |
| Publication status | Published - 30 Jan 2014 |
| Externally published | Yes |
Keywords
- Animals
- Cell Culture Techniques/instrumentation
- Cell Line
- Cells, Cultured
- Cerebral Cortex/metabolism
- Enzyme-Linked Immunosorbent Assay/methods
- Hippocampus/metabolism
- Mice
- Mice, Transgenic
- Nerve Growth Factors/metabolism
- Neurons/metabolism
- Phosphorylation
- Rats
- Receptor Protein-Tyrosine Kinases/metabolism
- Receptor, trkB/genetics