Table 1.
Summary of dye characteristics.
Figure 1.
Mean instantaneous D fit for different anti-EGFR Affibody conjugates.
Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
Figure 2.
Effect of logD and charge on affibody conjugate mobility.
Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 (A), and logD (B). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure 3.
Plots of distributions of mean instantaneous D fits for affibody-dye conjugates.
Dyes selected to represent high (Alexa 488), moderate (CF 633), low (Alexa 546), and very low (Atto 647N) spot mobility.
Figure 4.
Analysis of definitely mobile vs immobile or very slow moving spots.
A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm2/s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm2/s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values <0.1 µm2/s. Line shows linear regression fit to the data, R2 value indicating goodness of fit.
Figure 5.
Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody.
Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1st and 3rd quartile of the distributions.