Fig. 2

Optimizing a minimal tag for genetic code expansion (GCE)-based fluorescence labeling using α-tubulin. a Schematic view of the tags used and tested in b–e. Complete sequences of the tags are shown in Additional file 1: Figure S1c-f. b–e GCE-tags (specified in a) were sub-cloned to the N-terminal of α-tubulin using the pBUD-Pyl-RS-tub plasmid (Additional file 1: Figure S1a). HEK293T (b) or COS7 (c–e) cells were transfected with pBUD-Pyl-RS-tub plasmids carrying different tags, a WT version of α-tubulin, or without a target protein, and incubated for 48 h in the presence of the ncAA BCN-Lys. Cells were then subjected to western blot analysis (b) or labeled with SiR-Tet for 1 h and imaged live (c–e). Images in c are maximum intensity projections of 3D z-stacks obtained from representative cells. d Zoomed-in images of the regions marked in yellow squares in c. Intensity values along the red lines drawn in the images are plotted below, demonstrating the improvement in SNR. e SNR values measured in cells expressing α-tubulin with BCN-Lys incorporated either in position G45 or in the N-terminal via tags 3 or 4. The averaged SNR measured in cells expressing WT tubulin was 1.2 (red dashed line). The averaged SNR previously measured using GFP was 1.75 (green dashed line). n = 25. Results presented in b–e were obtained in at least 3 independent experiments. f Schematic view of the optimized GCE-tag used for further labeling. Scale bars: c 10 μm, d 2 μm