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Fig. 3
Titration of a low-affinity GFP binder modulates the mobility of extracellularly injected GFP in zebrafish embryos. (a) Different amounts of mRNA encoding the weak binder ( Kd = 600 nM in vitro) were injected into zebrafish embryos at the one-cell stage (50, 100, 200, or 400 pg); negative controls were left uninjected (0 pg of mRNA), and positive controls were injected with 50 pg of mRNA encoding the morphotrap ( Kd = 0.32 nM in vitro). Before the embryos were mounted for FRAP experiments at blastula stages, they were injected extracellularly with approximately 100 pg of recombinant GFP. FRAP experiments were performed as previously described(8,11) and analyzed using PyFRAP.(27) Scale bars correspond to 50 μm. (b and c) The effective diffusion coefficients ( Deff) of independent experiments executed as described for panel a are shown as black dots, and red lines indicate mean values. The dashed line in panel b shows an overlay with the effective diffusion model calculated from the equation in Figure 1d (see the Supporting Information for details).