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2.3. BRET2 assay
A BRET2 saturation assay was performed as previously described [7]. Briefly, 48 h after transfection, HEK293T S Tag transiently transfected with constant (1 μg) or increasing amounts (0.25–9 μg) of plasmids encoding for FGFR1Rluc and FGFR1GFP2, respectively, were rapidly washed twice in PBS, detached, and resuspended in the same buffer. For BRET2 measurement, coelenterazine-400a, also known as DeepBlue? C substrate, was added at a final concentration of 5 μM, and readings with the POLARstar Optima plate reader (BMG Labtechnologies, Offenburg, Germany).
For concentration–response and kinetic BRET2 experiments, HEK293T cells were transiently transfected at a constant ratio (1:2) of FGFR1Rluc/FGFR1GFP2 in presence of heparin (0.5 μM). Cells were treated with the indicated FGF ligand concentration or vehicle for 2 min before BRET2 measurement. FGF ligand-promoted BRET2 was calculated by subtracting the BRET2 ratio obtained in the absence of the FGF ligand from that obtained in the presence of the FGF ligand. In the case of kinetic measurements, coelenterazine-400a was added after the FGF ligand (30 ng/ml), just before BRET2 measurements. In each experiment, the specificities of FGFR1/FGFR1 interactions were assessed by comparison with cells expressing FGFR1GFP2 alone. As another negative control were used cells individually expressing FGFR1Rluc that were mixed prior to exposition to coelenterazine-400a with cells individually expressing FGFR1GFP2.