Figure 1
Principles of TR-FRET measurement of the protein level.
(a) Left: binding of two antibodies to the same protein molecule allow sufficient spatial proximity that enables fluorescence resonance energy transfer (FRET) between the donor and acceptor fluorophores conjugated to the antibodies. The signal is in proportion to the target protein level under tested experimental conditions. Right: the excitation light of TR-FRET experiments is given as 1-μs pulses and thus the background fluorescence from the media or the acceptor decay rapidly, whereas the fluorescence from the donor last for hundreds of μs, due to the unique property of the donor fluorophore made from lanthanides. When FRET occurs, the acceptor fluorescence lasts longer as well, due to the sustained energy transfer from the donor. A time window could then be selected to measure the FRET dependent signals, which are further calculated by the ratio between the intensity at 665 nm and the one at 615 nm to eliminate the influence from antibody-loading variations, etc. (b) A schematic picture of the epitopes targeted by the different antibodies used in this study.
