Fig. 6: Application of ReTagX for SRM.
a Illustration of ReTagX fused to the target protein. Created with BioRender.com. b Absorption and emission spectra of MaP655-TMP (10 μM) in the absence (orange line) and presence of ReTag1 (100 nM, pink line), ReTag2 (100 nM, blue line) and ReTag4 (100 nM, red line) after 1 h incubation. c Wash-free live-cell confocal images of co-cultured untransfected U2OS cells and U2OS H2B-GFP-ReTagX-expressing cells labeled with MaP655-TMP (200 nM) for 30 min. The untransfected cells are marked with white dashed lines. The white numbers indicate the average fluorescence intensity ratio between the nuclear signal (transfected cells) and the cytosol signal (normal cells) (Fnuc/Fcyt). d Statistical charts of nuclear brightness of TMP-probe in (c) (right, n = 23, 17, 19 cells, mean values ± S.E.M.). Not significant (n.s.), significant: p ≤ 0.0001 (****). e Wash-free wide field and SIM images of live U2OS cells expressing MitoNeet-ReTag4 stained with 200 nM MaP655-TMP. Scale bar: 5 μm. f Normalized fluorescence intensities of mitochondria plotted as a function of frame numbers in SIM images in U2OS cells expressing MitoNeet-ReTag1 and MitoNeet-ReTag4. n = three individual experiments, shadow indicates the SEM value. g Raw images of single ReTagX with different concentrations of MaP655-TMP (5 Hz). Scale bar: 10 μm. h Statistical charts of fluorescence intensity with background deduction for ReTagX (5 Hz, N = 100). Significant: p ≤ 0.0001 (****). i Representative fluorescence attenuation images of single ReTag1 (100 nM, orange line) and ReTag4 (100 nM, blue line, 500 nM, red line) (5 Hz). The dotted lines indicate the number of fluorophores on individual proteins. j Histogram showing the track-frame distribution of ReTag4 (8476 trajectory segments) and ReTag1(2786 trajectory segments) after fixed in coverslips. k Illustration of TNFR2 fused with either Halo-Tag or ReTag8. Created with BioRender.com. l Normalized single-molecule fluorescence intensities of TNFR2-Halo-Tag labeled with SiR-HTL (1 nM) and TNFR2-ReTag8 labeled with MaP655-TMP (200 nM) in live cells. Significant: p ≤ 0.0001 (****), n = 1495 and 1483 points from three independent experiments. m Representative trajectories of TNFR2-Halo-Tag labeled with SiR-HTL (1 nM) and TNFR2-ReTag8 with MaP655-TMP (200 nM) after 10 s and 50 s imaging, color coded by time. n Plot of the number of trajectories per 10 s interval for TNFR2 labeled with ReTag8, Halo-Tag, and TMP-Tag8. n = three individual experiments, error bar indicates the S.D. value. o Histogram showing the track-frame distribution of TNFR2-ReTag8 (2276 trajectories) and TNFR2-Halo-Tag (2222 trajectories) in live-cell imaging (20 Hz). Source data are provided as a Source Data file.
