Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells


Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Surface plasmon resonance microscopy.
Figure 2: Glycoprotein–lectin interaction.
Figure 3: Mapping glycoprotein distribution and binding kinetics.
Figure 4: Glycoprotein polarization during chemotaxis.
Figure 5: Mapping nAChRs.


  1. 1

    Cho, W. H. & Stahelin, R. V. Membrane–protein interactions in cell signaling and membrane trafficking. Annu. Rev. Biophys. Biomol. Struc. 34, 119–151 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Marinissen, M. J. & Gutkind, J. S. G-protein-coupled receptors and signaling networks: emerging paradigms. Trend. Pharm. Sci. 22, 368–376 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Hopkins, A. L. & Groom, C. R. The druggable genome. Nature Rev. Drug Disc. 1, 727–730 (2002).

    CAS  Article  Google Scholar 

  4. 4

    Adams, G. P. & Weiner, L. M. Monoclonal antibody therapy of cancer. Nature Biotech. 23, 1147–1157 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Rees, D. C., Congreve, M., Murray, C. W. & Carr, R. Fragment-based lead discovery. Nature Rev. Drug Disc. 3, 660–672 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Salamon, Z., Macleod, H. A. & Tollin, G. Surface plasmon resonance spectroscopy as a tool for investigating the biochemical and biophysical properties of membrane protein systems. 2. Applications to biological systems. Biochim. Biophys. Acta 1331, 131–152 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Lee, A. G. How lipids affect the activities of integral membrane proteins. Biochim. Biophys. Acta 1666, 62–87 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Fruh, V., Ijzerman, A. P. & Siegal, G. How to catch a membrane protein in action: a review of functional membrane protein immobilization strategies and their applications. Chem. Rev. 111, 640–656 (2011).

    Article  Google Scholar 

  9. 9

    Bally, M. et al. Liposome and lipid bilayer arrays towards biosensing applications. Small 6, 2481–2497 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Holden, M. A. et al. Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording. Nature Chem. Bio. 2, 314–318 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Dykstra, M. et al. Location is everything: lipid rafts and immune cell signaling. Annu. Rev. Immun. 21, 457–481 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Sato, T. K., Overduin, M. & Emr, S. D. Location, location, location: membrane targeting directed by PX domains. Science 294, 1881–1885 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Li, G. Y., Xi, N. & Wang, D. H. Probing membrane proteins using atomic force microscopy. J. Cell. Biochem. 97, 1191–1197 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Muller, D. J. & Engel, A. Atomic force microscopy and spectroscopy of native membrane proteins. Nature Protocols 2, 2191–2197 (2007).

    Article  Google Scholar 

  15. 15

    Groves, J. T., Parthasarathy, R. & Forstner, M. B. Fluorescence imaging of membrane dynamics. Annu. Rev. Biomed. Eng. 10, 311–338 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Schwarzenbacher, M. et al. Micropatterning for quantitative analysis of protein–protein interactions in living cells. Nature Methods 5, 1053–1060 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Johnson, A. E. Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes. Traffic 6, 1078–1092 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Wallrabe, H. & Periasamy, A. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotech. 16, 19–27 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Wang, W. et al. Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy. Nature Chem. 3, 249–255 (2011).

    Article  Google Scholar 

  21. 21

    Huang, B., Yu, F. & Zare, R. N. Surface plasmon resonance imaging using a high numerical aperture microscope objective. Anal. Chem. 79, 2979–2983 (2007).

    CAS  Article  Google Scholar 

  22. 22

    Kadurin, I. et al. Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b. Biochem. J. 412, 469–475 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Dell, A. & Morris, H. R. Glycoprotein structure determination mass spectrometry. Science 291, 2351–2356 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Durand, G. & Seta, N. Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin. Chem. 46, 795–805 (2000).

    CAS  PubMed  Google Scholar 

  25. 25

    Liu S. L. et al. Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot-based single-particle tracking. Biomaterials 32, 7616–7624 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Vila-Perello, M., Gallego, R. G. & Andreu, D. A simple approach to well-defined sugar-coated surfaces for interaction studies. ChemBioChem 6, 1831–1838 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Gingell, D., Todd, I. & Bailey, J. Topography of cell–glass apposition revealed by total internal reflection fluorescence of volume markers. J. Cell Biol. 100, 1334–1338 (1985).

    CAS  Article  Google Scholar 

  28. 28

    Sato, Y. et al. High mannose-binding lectin with preference for the cluster of 1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses. J. Biol. Chem. 286, 19446–19458 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Katrlik, J., Skrabana, R., Mislovicova, D. & Gemeiner, P. Binding of D-mannose-containing glycoproteins to D-mannose-specific lectins studied by surface plasmon resonance. Colloid Surf. A 382, 198–202 (2011).

    CAS  Article  Google Scholar 

  30. 30

    Rathanaswami, P., Babcook, J. & Gallo, M. High-affinity binding measurements of antibodies to cell-surface-expressed antigens. Anal. Biochem. 373, 52–60 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Troise, F. et al. Differential binding of human immunoagents and herceptin to the ErbB2 receptor. FEBS J. 275, 4967–4979 (2008).

    CAS  Article  Google Scholar 

  32. 32

    Lehmann, S. et al. An endogenous lectin and one of its neuronal glycoprotein ligands are involved in contact guidance of neuron migration. Proc. Natl Acad. Sci. USA 87, 6455–6459 (1990).

    CAS  Article  Google Scholar 

  33. 33

    Zieske, J. D., Higashijima, S. C. & Gipson, I. K. Con A-binding and WGA-binding glycoproteins of stationary and migratory corneal epithelium. Invest. Ophthalmol. Vis. Sci. 27, 1205–1210 (1986).

    CAS  PubMed  Google Scholar 

  34. 34

    Huppa, J. B. et al. TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Kataoka, M. & Tavassoli, M. Polarization of membrane-glycoproteins during monocyte chemotaxis. Exp. Cell Res. 153, 539–543 (1984).

    CAS  Article  Google Scholar 

  36. 36

    Russo, V. C. et al. Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation, migration, and invasion. Endocrinology 146, 4445–4455 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Albuquerque, E. X., Pereira, E. F. R., Alkondon, M. & Rogers, S. W. Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol. Rev. 89, 73–120 (2009).

    CAS  Article  Google Scholar 

  38. 38

    Eaton, J. B. et al. Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells. Mol. Pharmacol. 64, 1283–1294 (2003).

    CAS  Article  Google Scholar 

  39. 39

    DeFazio-Eli, L. et al. Quantitative assays for the measurement of HER1–HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action. Breast Cancer Res. 13, R44 (2011).

    CAS  Article  Google Scholar 

  40. 40

    Manz, B. N. & Groves, J. T. Spatial organization and signal transduction at intercellular junctions. Nature Rev. Mol. Cell Biol. 11, 342–352 (2010).

    CAS  Article  Google Scholar 

  41. 41

    Pick, H. et al. Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells. Biochemistry 42, 877–884 (2003).

    CAS  Article  Google Scholar 

Download references


We thank the National Institutes of Health (R21RR026235) for support.

Author information




W.W. and Y.Y. designed and performed the experiments. W.W., Y.Y., S.W., V.J.N. and N.J.T. discussed the results. Q.L. and J.W. provided the cell lines and helped with the immunofluorescence of nAChR. W.W. and N.J.T. wrote the paper. N.J.T. conceived the experiment and supervised the project.

Corresponding author

Correspondence to Nongjian Tao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 3071 kb)

Supplementary Movie 1

Supplementary Movie 1 (MOV 3660 kb)

Supplementary Movie 2

Supplementary Movie 2 (MOV 2698 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wang, W., Yang, Y., Wang, S. et al. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells. Nature Chem 4, 846–853 (2012).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing