Figure 4
From: Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
Auto-FPFA can detect subtle structural changes. Auto-FPFA was used to characterize V5V (red circles), V17V (blue squares), and V32V (green triangles). Each Venus dimers was composed of two Venus fluorophores separated by 5, 17, or 32 amino-acid linkers respectively. Auto-FPFA measurements of molecular brightness (a), diffusion time (τD, b), steady state anisotropy (c), and lifetime (d) of the three types of Venus dimers. For each Venus dimer sample three cell homogenates were prepared and five replicates were measured for each homogenate. Red bars indicate the mean, N = 15 (5 replicates of 3 homogenates for each dimer construct). Note the outlier that is easily detected in the V5V sample in panels c and d. We checked if the location on the micro-titer dish for this outlier was the same as the outlier previously observed in Fig. 3. It was not, indicating that it is unlikely that the outliers resulted from a systematic error in X/Y-axis positioning.
