Abstract
Here, we introduce a new quantitative FRET method (qs FRET) that allows for the measurement of apparent FRET efficiency and the concentrations of donor and acceptor-tagged membrane-bound proteins in cell-derived vesicles and live cells. This new method utilizes two-photon excitation and spectral imaging technology, where the complete emission spectrum of each pixel in an image is acquired. We utilize this method to measure the equilibrium association constant of fluorescent protein-tagged Receptor Tyrosine Kinases in live cells.
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