Comparison with FRET in the presence of a rigid linker (A) SDS-PAGE of the PCA/FRET probes with or without Fn7-8 domains. Lane 1, FKBP/Fn7-8/C; Lane 2, FRB/Ypet; Lane 3, FKBP/Cerulean; Lane 4 FKBP/Fn7-8/Cerulean. (B) FRET assays. Left: the assay using the probe pair without insertion. Right: the assay using the probe pair inserted with Fn7-8. (C) Fluc PCA using the probe inserted with Fn7-8. The probe/rapamycin concentrations of 750 nM (left) and 100 nM (right) were investigated (n = 3).