Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy

Chembiochem. 2021 Apr 6;22(7):1205-1209. doi: 10.1002/cbic.202000727. Epub 2020 Dec 17.


Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.

Keywords: Tub-tag labelling; antibody conjugates; immunofluorescence staining; nanobodies; peptide nucleic acids; super-resolution microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemistry
  • Azides / chemistry
  • Catalysis
  • Cell Line
  • Copper / chemistry
  • Cycloaddition Reaction
  • DNA / chemistry*
  • Green Fluorescent Proteins / chemistry
  • Humans
  • Immunoconjugates / chemistry
  • Immunoconjugates / metabolism
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / metabolism
  • Microscopy, Fluorescence*
  • Peptide Nucleic Acids / chemistry*
  • Single-Domain Antibodies / chemistry


  • Alkynes
  • Azides
  • Immunoconjugates
  • Immunoglobulin Fragments
  • Peptide Nucleic Acids
  • Single-Domain Antibodies
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Copper
  • DNA