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Targeting and tracing antigens in live cells with fluorescent nanobodies

Nature Methods volume 3, pages 887889 (2006) | Download Citation

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Abstract

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

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Acknowledgements

We thank R.Y. Tsien, J. Ellenberg and K.F. Sullivan for providing expression vectors and cell lines, and J. Füner for the alpaca immunization. This work was supported by grants from the Deutsche Forschungsgemeinschaft to M.C.C. and H.L.

Author information

Author notes

    • Ulrich Rothbauer
    •  & Kourosh Zolghadr

    These authors contributed equally to this work.

Affiliations

  1. Ludwig Maximilians University Munich, Department of Biology II, Grosshaderner Str. 2, 82152 Planegg-Martinsried, Germany.

    • Ulrich Rothbauer
    • , Kourosh Zolghadr
    • , Lothar Schermelleh
    •  & Heinrich Leonhardt
  2. Institute of Gene Biology of the Russian Academy of Sciences, Vavilov Str. 34/5, 119334 Moscow, Russia.

    • Sergei Tillib
  3. Max Delbrueck Center for Molecular Medicine, Robert-Roessle Str. 10, 13125 Berlin, Germany.

    • Danny Nowak
    • , Anja Gahl
    •  & M Cristina Cardoso
  4. Vrije Universiteit Brussel, Laboratory of Cellular and Molecular Immunology, Pleinlaan 2, 1050 Brussels, Belgium.

    • Natalija Backmann
    • , Katja Conrath
    •  & Serge Muyldermans

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Heinrich Leonhardt.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Schematic comparison of a conventional IgG and a heavy-chain IgG from Camelidae and various antigen-binding fragments derived thereof.

  2. 2.

    Supplementary Fig. 2

    The GFP—chromobody is present as a stable monomeric protein in mammalian cells.

  3. 3.

    Supplementary Fig. 3

    Schematic representation of the fusion proteins used in this study.

  4. 4.

    Supplementary Fig. 4

    Antigen tracing with chromobodies.

  5. 5.

    Supplementary Methods

Videos

  1. 1.

    Supplementary Video 1

    Tracing of a chromatin protein throughout mitosis. Time lapse imaging of a HeLa cell stably expressing histone H2B-GFP transfected with GFP—chromobody. At the time imaging was started (0 h), this cell was in late G2 phase.

  2. 2.

    Supplementary Video 2

    Tracing of a component of the replication machinery throughout S phase until G2. Time lapse imaging of cotransfected HeLa cells expressing GFP—PCNA and GFP—chromobody. At the start of imaging (0 h), the cells were in early to mid S phase.

About this article

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DOI

https://doi.org/10.1038/nmeth953

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