Jens Randel Nyengaard

Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR

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Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR. / Skeldal, Sune; Kjaergaard, Maj M.; Alwasel, Saleh; Nyengaard, Jens R.

In: International Journal of Biochemistry and Molecular Biology, Vol. 6, No. 2, 01.01.2015, p. 17-25.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Skeldal, S, Kjaergaard, MM, Alwasel, S & Nyengaard, JR 2015, 'Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR', International Journal of Biochemistry and Molecular Biology, vol. 6, no. 2, pp. 17-25.

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Author

Skeldal, Sune ; Kjaergaard, Maj M. ; Alwasel, Saleh ; Nyengaard, Jens R. / Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR. In: International Journal of Biochemistry and Molecular Biology. 2015 ; Vol. 6, No. 2. pp. 17-25.

Bibtex

@article{dd86de5a5c594efb8130b8d147818331,
title = "Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR",
abstract = "Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.",
keywords = "FRET-based fluorescence plate reader assay, High throughput, P75, ProNGF, Sortilin",
author = "Sune Skeldal and Kjaergaard, {Maj M.} and Saleh Alwasel and Nyengaard, {Jens R.}",
year = "2015",
month = jan,
day = "1",
language = "English",
volume = "6",
pages = "17--25",
journal = "International Journal of Biochemistry and Molecular Biology",
issn = "2152-4114",
publisher = "E-Century Publishing Corporation",
number = "2",

}

RIS

TY - JOUR

T1 - Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR

AU - Skeldal, Sune

AU - Kjaergaard, Maj M.

AU - Alwasel, Saleh

AU - Nyengaard, Jens R.

PY - 2015/1/1

Y1 - 2015/1/1

N2 - Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.

AB - Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.

KW - FRET-based fluorescence plate reader assay

KW - High throughput

KW - P75

KW - ProNGF

KW - Sortilin

UR - http://www.scopus.com/inward/record.url?scp=84958173779&partnerID=8YFLogxK

M3 - Journal article

AN - SCOPUS:84958173779

VL - 6

SP - 17

EP - 25

JO - International Journal of Biochemistry and Molecular Biology

JF - International Journal of Biochemistry and Molecular Biology

SN - 2152-4114

IS - 2

ER -