Patent ID: 8470523
Filing Date: 2013-06-25
Classification: G01N

Abstract:
1. A method of detecting interactions between intracellular biomolecules or a translocation or conformational change of intracellular biomolecules in living cells, comprising the following steps: a) labeling a first biomolecule, in a living cell, with a first fluorescent compound having a long fluorescence lifetime, selected from the group consisting of a rare earth chelate and a rare earth cryptate, to produce a first fluorescently labeled biomolecule; b) labeling at least one second biomolecule, in said living cell, with a second fluorescent compound, to produce a second fluorescently labeled biomolecule; c) subjecting the living cells to a specific stimulation adapted to the biological response to be studied, in the presence or absence of a compound belonging to a bank of test molecules; d) subjecting the living cells to a light source whose wavelength excites said first long-lived fluorescent compound; e) measuring the intensity of the fluorescence emitted by said first and second fluorescent compounds; f) calculating the ratio between the fluorescence intensities of said first fluorescent compound and said second fluorescent compound, or measuring the lifetime of the first or second fluorescent compound; and g) comparing the measured signals with those obtained before stimulation of the cells, wherein said first and at least second fluorescent compounds are time-resolved FRET (TR-FRET) partners; said first and/or the second biomolecule are each expressed as a fusion protein with a suicide enzyme, and step a) and/or b) are achieved by extracellularly contacting the cell with a substrate for the suicide enzyme, said substrate being covalently bonded to the first or second fluorescent compound, and at least one of the fluorescent compounds