Utilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells
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Utilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells. / Antila, Hanna; Autio, Henri; Turunen, Laura; Harju, Kirsi; Tammela, Päivi; Wennerberg, Krister; Yli-Kauhaluoma, Jari; Huttunen, Henri J; Castrén, Eero; Rantamäki, Tomi.
In: Journal of Neuroscience Methods, Vol. 222, 30.01.2014, p. 142-6.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Utilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells
AU - Antila, Hanna
AU - Autio, Henri
AU - Turunen, Laura
AU - Harju, Kirsi
AU - Tammela, Päivi
AU - Wennerberg, Krister
AU - Yli-Kauhaluoma, Jari
AU - Huttunen, Henri J
AU - Castrén, Eero
AU - Rantamäki, Tomi
N1 - Copyright © 2013 Elsevier B.V. All rights reserved.
PY - 2014/1/30
Y1 - 2014/1/30
N2 - 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.
AB - 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.
KW - Animals
KW - Cell Culture Techniques/instrumentation
KW - Cell Line
KW - Cells, Cultured
KW - Cerebral Cortex/metabolism
KW - Enzyme-Linked Immunosorbent Assay/methods
KW - Hippocampus/metabolism
KW - Mice
KW - Mice, Transgenic
KW - Nerve Growth Factors/metabolism
KW - Neurons/metabolism
KW - Phosphorylation
KW - Rats
KW - Receptor Protein-Tyrosine Kinases/metabolism
KW - Receptor, trkB/genetics
U2 - 10.1016/j.jneumeth.2013.11.001
DO - 10.1016/j.jneumeth.2013.11.001
M3 - Journal article
C2 - 24239780
VL - 222
SP - 142
EP - 146
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
ER -
ID: 199431436