Protocol | Published:

A general protocol for the generation of Nanobodies for structural biology

Nature Protocols volume 9, pages 674693 (2014) | Download Citation


There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo–matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6–12 months.

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We thank members and associates of the Steyaert, Muyldermans, Hol and Kobilka laboratories, past and present, for their assorted contributions over the years to this work. In particular, we acknowledge the contributions of N. Buys and Y.J. Park. The Steyaert laboratory was supported by the Fonds Wetenschappelijk Onderzoek-Vlaanderen through research grants G011110N and G049512N, Innoviris Brussels through the Impulse Life Science program BRGEOZ132, the Belgian Federal Science Policy Office through IAP7-40 and by the SBO program IWT120026 from the Flemish Agency for Innovation by Science and Technology. B.K.K. received support from US National Institutes of Health (NIH) grants R01NS028471 and R01GM083118 and from the Mathers Foundation. The research in the laboratory of W.G.J.H. was supported by the National Institute of Allergy and Infectious Diseases (NIAID) and the National Institute of General Medical Sciences (NIGMS) of the NIH under award numbers AI34501 and GM077418. S.T. received a doctoral fellowship from the Fonds Wetenschappelijk Onderzoek-Vlaanderen. S.G.F.R is supported by the Lundbeck Foundation.

Author information


  1. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

    • Els Pardon
    • , Toon Laeremans
    • , Sarah Triest
    • , Alexandre Wohlkönig
    •  & Jan Steyaert
  2. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.

    • Els Pardon
    • , Toon Laeremans
    • , Sarah Triest
    • , Alexandre Wohlkönig
    • , Serge Muyldermans
    •  & Jan Steyaert
  3. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

    • Søren G F Rasmussen
  4. Pharma Research and Early Development (pRED), Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche, Basel, Switzerland.

    • Armin Ruf
  5. Cellular and Molecular Immunology, VUB, Brussels, Belgium.

    • Serge Muyldermans
  6. Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, Seattle, Washington, USA.

    • Wim G J Hol
  7. Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, California, USA.

    • Brian K Kobilka


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J.S. developed the concept of Nanobody-assisted crystallography in collaboration with W.G.J.H. and B.K.K.; E.P., T.L., S.T., A.W. and J.S. worked out the protocol. E.P., T.L., S.M., W.G.J.H., B.K.K. and J.S. contributed to the Introduction. E.P., T.L., S.G.F.R., A.R., B.K.K. and J.S. performed the experiments described in the Anticipated Results and all authors participated in discussions on technical and conceptual aspects of the protocol and the editing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jan Steyaert.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Figure 1

    Strategies to amplify the Nanobody repertoire by PCR from PBL cDNA.

  2. 2.

    Supplementary Figure 2

    Map and sequence information for phage display vector pMES4.

  3. 3.

    Supplementary Figure 3

    Map and sequence information for phage display vector pMESy4.

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