Fig. 4: Quantitation of intasome-LEDGF/p75 stoichiometry by single-molecule TIRF microscopy.

a Schematic of the photobleaching experiment. Intasomes containing biotin- and Cy3-conjugated vDNA were immobilized on a streptavidin-coated cover slip (light blue rectangle). IN and vDNA are shown as gray and black cartoons, respectively; biotin and Cy3 are depicted as a purple circle and yellow solar symbol, respectively. Following incubation with LEDGF/p75 (red cartoons) conjugated with Surf649 (red solar symbol) and a wash with buffer containing 0.2, 0.5, or 1 M NaCl, immobilized intasomes were observed by TIRF microscopy. The individual steps of Surf649 photobleaching during illumination with a 640-nm laser (hν) were counted. b Representative images of surface attached intasome (vDNA-Cy3, yellow; LEDGF/p75-Surf649, red) molecules. The field of view of 14.4 by 14.4 μm shown here is representative of the dataset, which included three areas of 81.9 by 81.9 μm (see Methods section for details). Dotted, white line squares indicate individual intasome-LEDGF/p75 complexes. c Left: examples of stepwise photobleaching traces of LEDGF/p75-Surf649 at increasing NaCl concentration: 0.2 M (blue), 0.5 M (magenta), and 1.0 M (dark gray). The vertical axis represents fluorescence in arbitrary units (arb. units). Right: Box-and-whiskers plots summarizing statistical analysis of the number of LEDGF/p75-Surf649 photobleaching steps per intasome, for various NaCl concentrations (see Supplementary Table 2 for further details). Each box encloses data between 25th and 75th percentiles, with the median value displayed as a horizontal line. Vertical lines (whiskers) indicate 10th and 90th percentiles; outliers are indicated with closed circles. Source data are provided as a Source Data file.