2-hybrid 3-cube FRET experiments were carried out with standard protocols similarly shared by several groups20 (link),24 ,56 (link). Briefly, experiments were performed on an inverted epi-fluorescence microscope (Ti-U, Nikon), with computer-controlled filter wheels (Sutter Instrument) to coordinate with diachronic mirrors for appropriate imaging at excitation, emission, and FRET channels. The filters used in the experiments were excitation: 438/24 (FF01-438/24-25, Semrock) and 480/30 (FITC, Nikon); emission: 483/32 (FF01-483/32-25, Semrock) and 535/40 (FITC, Nikon); dichroic mirrors: 458 nm (FF458-Di02-25 × 36, Semrock) and 505 nm (FITC, Nikon). Fluorescence images were acquired by Neo sCMOS camera (Andor Technology) and analyzed with 33-FRET algorithms coded in Matlab (Mathworks), mainly based on the following formula:
FRmax represents the maximum FRET ratio, and Dfree denotes the equivalent free donor (CFP-tagged) concentration. Kd (effective dissociation equilibrium constant) is calculated from an iterative procedure to evaluate the binding affinity for each pair of binding partners. FRET imaging experiments were performed with HEK293 cells in Tyrode’s buffer containing 2 mM Ca2+.
FRmax represents the maximum FRET ratio, and Dfree denotes the equivalent free donor (CFP-tagged) concentration. Kd (effective dissociation equilibrium constant) is calculated from an iterative procedure to evaluate the binding affinity for each pair of binding partners. FRET imaging experiments were performed with HEK293 cells in Tyrode’s buffer containing 2 mM Ca2+.
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