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Single-molecule recognition force spectroscopy of transmembrane transporters on living cells

Nature Protocols volume 6, pages 1443–1452 (2011)Cite this article

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Abstract

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.

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Figure 1: AFM force-distance cycle.
Figure 2: Linkage of ligands to AFM tips.
Figure 3: Positioning of AFM tip.
Figure 4: Cell images of living G6D3 cells.
Figure 5: AFM images of fixed G6D3 cells.
Figure 6: Recognition of SGLT1 on the surface of intact cells by AFM tip coated with specific antibodies (PAN3-2 antibodies).
Figure 7: Recognition of SGLT1 by a 1-thio-D-glucose-coated AFM tip.

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Acknowledgements

We acknowledge the support by the Austrian Science Foundation, the Max Planck Institute and the Faculty of Science, Mahidol University. We also thank the National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Ministry of Science and Technology, Thailand, through its program of Center of Excellence Network. We thank L. Wildling and H. J. Gruber for their expertise in tip chemistry. Help from C. Rankl and all collaborators in Dortmund is gratefully acknowledged.

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Authors and Affiliations

  1. Institute for Biophysics, Johannes Kepler University of Linz, Linz, Austria

    Theeraporn Puntheeranurak, Isabel Neundlinger & Peter Hinterdorfer

  2. Department of Biology, Faculty of Science, Mahidol University and NANOTEC Center of Excellence at Mahidol University, Bangkok, Thailand

    Theeraporn Puntheeranurak

  3. Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Rolf K H Kinne

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  1. Theeraporn Puntheeranurak
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Contributions

T.P. designed and conducted the experiments, analyzed data and wrote the manuscript; I.N. commented on the manuscript; R.K.H.K. and P.H. designed, discussed and edited the manuscript.

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Correspondence to Peter Hinterdorfer.

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Puntheeranurak, T., Neundlinger, I., Kinne, R. et al. Single-molecule recognition force spectroscopy of transmembrane transporters on living cells. Nat Protoc 6, 1443–1452 (2011). https://doi.org/10.1038/nprot.2011.370

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