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Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments

Nature Protocols volume 5, pages 975–985 (2010)Cite this article

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Abstract

The success of single-molecule (SM) experiments critically depends on the functional immobilization of the biomolecule(s) to be studied. With the continuing trend of combining SM fluorescence with SM force experiments, methods are required that are suitable for both types of measurements. We describe a general protocol for the site-specific and covalent coupling of any type of biomolecule that can be prepared with a free thiol group. The protocol uses a poly(ethylene glycol) (PEG) spacer, which carries an N-hydroxy succinimide (NHS) group on one end and a maleimide group on the other. After reacting the NHS group with an amino-functionalized surface, the relatively stable but highly reactive maleimide group allows the coupling of the biomolecule. This protocol provides surfaces with low fluorescence background, low nonspecific binding and a large number of reactive sites. Surfaces containing immobilized biomolecules can be obtained within 6 h.

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Figure 1: An overview of the immobilization procedure.
Figure 2: An overview of the protocol.
Figure 3: An example of an experiment carried out to verify the site-specific immobilization of an enzyme.

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Acknowledgements

We thank Hermann E. Gaub and all members of the Gaub lab for using the protocol in various SM experiments and for many valuable discussions leading to continuous improvements of the protocol. This work was supported by the German Research Foundation (DFG) and the European Union.

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Authors and Affiliations

  1. Lehrstuhl für Angewandte Physik & Center for Nanoscience, LMU München, München, Germany

    Julia L Zimmermann, Thomas Nicolaus, Gregor Neuert & Kerstin Blank

  2. Max-Planck-Institut für extraterrestrische Physik, Garching, Germany

    Julia L Zimmermann

  3. Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Gregor Neuert

  4. Department of Molecular Materials, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands

    Kerstin Blank

Authors
  1. Julia L Zimmermann
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Contributions

T.N. and G.N. developed the silanization protocols; J.L.Z. and K.B. developed the coupling procedures for the biomolecules; G.N. and J.L.Z. carried out SM experiments; and J.L.Z. and K.B. wrote the paper.

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Correspondence to Kerstin Blank.

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The authors declare no competing financial interests.

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Zimmermann, J., Nicolaus, T., Neuert, G. et al. Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments. Nat Protoc 5, 975–985 (2010). https://doi.org/10.1038/nprot.2010.49

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