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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  • Sune Skeldal
  • Maj M. Kjaergaard, The Lundbeck Foundation Research Center MIND, Stereology and Electron Microscopy Laboratory and Centre for Stochastic Geometry and Advanced Bioimaging
  • ,
  • Saleh Alwasel, King Saud University, Riyadh
  • ,
  • Jens R. Nyengaard

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.

OriginalsprogEngelsk
TidsskriftInternational Journal of Biochemistry and Molecular Biology
Vol/bind6
Nummer2
Sider (fra-til)17-25
Antal sider9
ISSN2152-4114
StatusUdgivet - 1 jan. 2015

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