Fig. 7.

Qdot conjugation strategies and data analysis. (A) Streptavidin (red)-coated quantum dot (green) is conjugated to His-tagged Rad4–Rad23 via the biotin-conjugated mouse-anti-His antibody (gray). (B) His-tagged Rad4–Rad is labeled by goat-antirabbit secondary antibody (wheat)-conjugated quantum dot (red) via a rabbit-anti-His primary antibody (purple). (C) Top: Representative kymograph of a diffusing particle. Middle: Plot of position, in the units of pixels (1 pixel = 46 nm), vs time, after fitting the light intensity profile at each time point in the kymograph with a one-dimensional Gaussian. Bottom: Plot of mean squared displacement (MSD), calculated from Gaussian-fitted positions, vs time steps. Orange dashed line is the result of fitting the initial portion of the MSD curve to the equation MSD = 2Dt^α. Inset: three types of one-dimensional diffusion characterized by different α values: superdiffusion (red), random diffusion (blue), and subdiffusion (green). Based on Movie 1 in the online version at http://dx.doi.org/10.1016/bs.mie.2017.03.027. Adapted with permission from Kong, M., & Van Houten, B. (2016). Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins. Progress in Biophysics and Molecular Biology. http://dx.doi.org/10.1016/j.pbiomolbio.2016.12.004 (fig. 2C).