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.


METHODS
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.


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.
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland.
Hanna
H
Antila
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland.
Henri
H
Autio
Institute For Molecular Medicine Finland (FIMM), P.O. Box 20, Tukholmankatu 8, FI-00014 University of Helsinki, Finland.
Laura
L
Turunen
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, Viikinkaari 5 E, FI-00014 University of Helsinki, Finland.
Kirsi
K
Harju
Centre for Drug Research, Faculty of Pharmacy, P.O. Box 56, Viikinkaari 5 E, FI-00014 University of Helsinki, Finland.
Päivi
P
Tammela
Institute For Molecular Medicine Finland (FIMM), P.O. Box 20, Tukholmankatu 8, FI-00014 University of Helsinki, Finland.
Krister
K
Wennerberg
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, Viikinkaari 5 E, FI-00014 University of Helsinki, Finland.
Jari
J
Yli-Kauhaluoma
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland; Hermo Pharma Ltd., Viikinkaari 4, FI-00790 Helsinki, Finland.
Henri J
HJ
Huttunen
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland.
Eero
E
Castrén
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland. Electronic address: tomi.rantamaki@helsinki.fi.
Tomi
T
Rantamäki
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006624:Hippocampus / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009414:Nerve Growth Factors / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D020813:Receptor, trkB / Q000235:genetics / Q000378:metabolism
D009414:Nerve Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D020813:Receptor, trkB
BDNF; ELISA; In situ; TrkB phosphorylation