Fig. 2

Detection of ALFA-tagged target proteins by fluorescent Western blot. a Lysates from COS-7 cells transfected with ALFA-vimentin or a mock control plasmid were analyzed by SDS–PAGE and Western blot. The blot was developed with NbALFA directly coupled to IRDye800CW or a mouse anti-tubulin primary antibody followed by FluoTag-X2 anti-Mouse IRDye680RD. Complete lanes including molecular weight markers are shown in Supplementary Fig. 4a. b Sketch of the E. coli maltose-binding protein (MBP) simultaneously fused to FLAG®-tag (FLAG), HA-tag (HA), myc-tag (myc) and ALFA-tag (ALFA). The multi-tag fusion protein was used for the experiments shown in c, d. ALFA-tag is shown in blue. c Dilution series of the protein sketched in b were spotted onto nitrocellulose membranes. Established monoclonal antibodies (M2, 9E10 and F-7) were used together with a goat anti-mouse secondary antibody coupled IRDye800CW to detect the FLAG®-, myc- and HA-tags, respectively. The ALFA-tag was detected using NbALFA coupled to IRDye800CW. The complete experiment with controls is shown in Supplementary Fig. 4b. d Double-logarithmic plot showing quantification of signals obtained in c in arbitrary units (a.u.) versus the amount of spotted target protein. Lines represent linear fits to the obtained values. Even without signal amplification by a secondary antibody, signals obtained using NbALFA were 3- to > 10-times stronger than by established reagents recognizing the other epitope tags. At the same time, detection with NbALFA was 10-fold more sensitive and showed an excellent linearity over approximately three orders of magnitude