Extended Data Figure 3 : smFRET experimental controls.

From: Single-molecule analysis of ligand efficacy in β2AR–G-protein activation

Extended Data Figure 3

a, Site-specific labelling. SDS–PAGE gels under green (540 nm) or near infrared (740 nm) illumination for fluorescence visualization of Cy3B* or Cy7* labelling of β2∆6 and β2∆6-148C/266C. Coomassie-stained gel image is shown as a gel-loading control. Digestion with factor Xa protease and deglycosylation with PNGase F leads to separation of the 148C and 266C labelling sites on generated N-terminal and C-terminal fragments, respectively. For gel source data, see Supplementary Fig. 1. b, Quantification of Cy3B*/Cy7*-labelling specificity of full-length β2∆6-148C/266C. Data are normalized to β2∆6-148C/266C labelling. c, Specificity of streptavidin-mediated receptor immobilization. Frame capture from immobilization movies showing labelled β2∆6-148C/266C on streptavidin-free (−SA) or streptavidin-coated (+SA) surfaces. Bar graph shows the number of immobilized, labelled β2AR in these conditions. Error bars represent the s.d. from two replicates. d, Schematic of labelled β2AR immobilization via biotinylated alprenolol (alp-biotin). e, FRET population contour plot and histogram for alp-biotin-immobilized receptor shows correspondence with the FRET population distribution of biotin-M1-Fab-immobilized, alprenolol-bound, labelled β2AR (Fig. 2b). Histogram error bars represent the s.d. from four replicates, with n total molecules analysed. f, FRET population contour plots (top) and histograms (bottom) for adrenaline titration on biotin-M1-Fab-immobilized, labelled β2∆6-148C/266C (Fig. 2c). Dashed lines (blue) highlight the mean FRET values for the lowest (2 nM; top dashed line) and highest (200 μM; bottom dashed line) adrenaline concentrations tested. Histogram error bars represent the s.d. from three replicates with n total molecules analysed. The scale bar on the right indicates relative populations for the contour plots.