A general protocol for the generation of Nanobodies for structural biology

Nat Protoc. 2014 Mar;9(3):674-93. doi: 10.1038/nprot.2014.039. Epub 2014 Feb 27.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotechnology / methods*
  • Camelus
  • Cell Surface Display Techniques
  • Cloning, Molecular
  • Crystallization / methods*
  • DNA Primers / genetics
  • Models, Molecular*
  • Protein Conformation*
  • Single-Domain Antibodies / biosynthesis*


  • DNA Primers
  • Single-Domain Antibodies