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. 2017 Apr 11;112(7):1316–1327. doi: 10.1016/j.bpj.2017.02.028

Figure 1.

Figure 1

Single-molecule tracking of overexpressed LacI-PAmCherry and LacIMut-PAmCherry. We use tracking PALM to observe single LacI-PAmCherry molecules by photoactivating, then imaging using 15-ms exposures. (A) An example cell with all LacI localizations. (B) The same cell showing LacI tracks with four or more steps, classified as bound (red) and mobile (blue) by thresholding of their apparent diffusion, D∗. (C) D∗ histogram of the full-length LacI with ONPG (which was added to outcompete IPTG used to overexpress the LacI) fit with two species and three species (inset). (D) D∗ histogram for LacI without the DNA-binding domain fit with three species and two species (inset). Without the DNA-binding domain, LacIMut shows a reduced bound (or slow) fraction of molecules. (E). After IPTG is added, LacI shows reduced specific and non-specific binding to DNA. The three-species fit is shown together with the two-species fit (inset). (F) Addition of 1 mM IPTG to LacIMut does not change significantly its diffusion profile.