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J Neurosci Methods. 2014 Jan 30;222:142-6. doi: 10.1016/j.jneumeth.2013.11.001. Epub 2013 Nov 12.

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

Author information

1
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland.
2
Institute For Molecular Medicine Finland (FIMM), P.O. Box 20, Tukholmankatu 8, FI-00014 University of Helsinki, Finland.
3
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, Viikinkaari 5 E, FI-00014 University of Helsinki, Finland.
4
Centre for Drug Research, Faculty of Pharmacy, P.O. Box 56, Viikinkaari 5 E, FI-00014 University of Helsinki, Finland.
5
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland; Hermo Pharma Ltd., Viikinkaari 4, FI-00790 Helsinki, Finland.
6
Neuroscience Center, P.O. Box 56, Viikinkaari 4, FI-00014 University of Helsinki, Finland. Electronic address: tomi.rantamaki@helsinki.fi.

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.

KEYWORDS:

BDNF; ELISA; In situ; TrkB phosphorylation

PMID:
24239780
DOI:
10.1016/j.jneumeth.2013.11.001
[Indexed for MEDLINE]

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