Byrne, R.D., Larijani, B. and Poccia, D.L.
2016 Methods in Molecular Biology 1411: in press.
In, The Nuclear Envelope: Methods and Protocols
FRET–FLIM techniques have wide application in the study of protein and protein–lipid interactions in cells.
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.
We have used this 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.