Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

  1. Tino Pleiner
  2. Mark Bates
  3. Sergei Trakhanov
  4. Chung-Tien Lee
  5. Jan Erik Schliep
  6. Hema Chug
  7. Marc Böhning
  8. Holger Stark
  9. Henning Urlaub
  10. Dirk Görlich  Is a corresponding author
  1. Max Planck Institute for Biophysical Chemistry, Germany
  2. Max Planck Institute for Biophysical Chemistry,, Germany

Abstract

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks - each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

Article and author information

Author details

  1. Tino Pleiner

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Mark Bates

    Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Sergei Trakhanov

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Chung-Tien Lee

    Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Jan Erik Schliep

    3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Hema Chug

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Marc Böhning

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Holger Stark

    3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry,, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Henning Urlaub

    Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Dirk Görlich

    Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    For correspondence
    goerlich@mpibpc.mpg.de
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Karsten Weis, ETH Zürich, Switzerland

Publication history

  1. Received: September 3, 2015
  2. Accepted: December 2, 2015
  3. Accepted Manuscript published: December 3, 2015 (version 1)
  4. Accepted Manuscript updated: December 5, 2015 (version 2)
  5. Version of Record published: February 3, 2016 (version 3)
  6. Version of Record updated: March 16, 2016 (version 4)

Copyright

© 2015, Pleiner et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Tino Pleiner
  2. Mark Bates
  3. Sergei Trakhanov
  4. Chung-Tien Lee
  5. Jan Erik Schliep
  6. Hema Chug
  7. Marc Böhning
  8. Holger Stark
  9. Henning Urlaub
  10. Dirk Görlich
(2015)
Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation
eLife 4:e11349.
https://doi.org/10.7554/eLife.11349
  1. Further reading

Further reading

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