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Methods Mol Biol. 2016;1411:123-32. doi: 10.1007/978-1-4939-3530-7_7.

The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

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Signalling Programme, The Babraham Institute, Cambridge, UK.
Cell Biophysics Laboratory, Ikerbasque Basque Foundation for Science, Unidad de Biofísica (CSIC UPV/EHU), Leioa, Bizkaia, Spain.
Research Centre for Experimental Marine Biology and Biotechnology (PiE), University of the Basque Country (UPV), Leioa, Bizkaia, Spain.
Department of Biology, Amherst College, 324 McGuire Life Sciences Building, P.O. Box: AC# 2237, Amherst, MA, 01002, USA.


FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.


Antibody; FRET; Membrane fusion; Nuclear envelope; Two-photon FLIM

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