Fig. 1
From: The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
Nanobody-based detection of ALFA-tagged proteins in immunofluorescence applications. a Sketch of NbALFA bound to the ALFA-tag. Given are ALFA sequences for tagging at various positions. b Sequence of NbALFA. Gray boxes indicate CDRs 1–3 (complementarity determining regions 1–3; AbM definition). c COS-7 cells transfected with Tom70-EGFP-ALFA were fixed with paraformaldehyde (PFA) and stained with NbALFA coupled to AbberiorStar635P (NbALFA-Ab635P). Left to right: NbALFA-Ab635P; intrinsic EGFP signal; overlay incl. DAPI stain; sketch illustrating the detection of Tom70-EGFP-ALFA. Scale bars: 20 µm. d ALFA-vimentin was detected with NbALFA-Ab635P after fixation with 4% PFA, 2% glutaraldehyde (GA), or 100% Methanol (MeOH). Scale bars: 20 µm. e STED and confocal images of COS-7 cell transiently transfected with ALFA-vimentin and stained with NbALFA-Ab635P. Color scheme: Red Hot (ImageJ). Scale bars: 2.5 µm. f HeLa cells transfected with ALFA-vimentin were stained with NbALFA bearing a 10-nucleotide single stranded DNA before imaging by 3D DNA-PAINT. Scale bars: 2.5 µm. The histogram refers to a region (small yellow rectangle) where 2 vimentin filaments are resolved although being only ~90 nm apart. The localization precision was 5.2 nm. g COS-7 cells were co-transfected with an NbALFA-mScarlet-I fusion and ALFA-vimentin. NbALFA-mScarlet-I co-localizes with ALFA-vimentin detected by immunofluorescence using NbALFA-Ab635P. This shows that NbALFA expressed in the cytoplasm of mammalian cells can be used for targeting ALFA-tagged proteins in living cells. Scale bars: 20 µm. N: N-terminus; C: C-terminus, TM: transmembrane domain. Colors scheme used for sketches: NbALFA (orange), ALFA-tag (blue), GFP (green), mScarlet-I (red), fluorophore (red star), FLAG®-tag (gray), vimentin (light blue)
