Figure 3.

Saturation binding of fluorescently (HiLyte^TM Fluor 488) labelled EGFR nanobodies (A) Q44c-HL488 and (B) Q86c-HL488 in absence (closed circles) or presence (open circles) of 100 nM EGF. NanoBRET experiments were performed using full-length N-terminal nanoluciferase-EGFR stably expressing HEK293 cells. Nanobodies and EGF were added simultaneously and incubated for 30 minutes at 37^oC. Experiments were performed in HBSS containing 0.2 % BSA. The NLuc substrate furimazine (12.5 nM) was added and plates incubated for 5 minutes then luminescence and fluorescence emissions were measured using a BMG Pherastar. Data are combined mean ± SEM from five independent experiments, where each experiment was performed in triplicate.