Skip to main content

Thank you for visiting nature.com. 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.

  • Protocol
  • Published:

Labeling proteins on live mammalian cells using click chemistry

Nature Protocols volume 10, pages 780–791 (2015)Cite this article

Subjects

Abstract

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron–demand Diels–Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced—by site-directed mutagenesis—at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4–7 d, followed by the described transfections and labeling reaction steps, which can take 3–4 d.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the ncAA and the dye derivatives used in the protocol.
Figure 2: Optimization of Amber mutant expression in mammalian cells for subsequent labeling.
Figure 3: SPAAC labeling of IR.
Figure 4: SPIEDAC labeling of IR with H-Tet-Cy5.
Figure 5: SPIEDAC labeling of IR with Me-Tet-Cy5.

Similar content being viewed by others

References

  1. Glaeser, J., Nuss, A.M., Berghoff, B.A. & Klug, G. Singlet oxygen stress in microorganisms. Adv. Microb. Physiol. 58, 141–173 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Chudakov, D.M., Matz, M.V., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90, 1103–1163 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Fernandez-Suarez, M. & Ting, A.Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929–943 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. van de Linde, S., Heilemann, M. & Sauer, M. Live-cell super-resolution imaging with synthetic fluorophores. Annu. Rev. Phys. Chem. 63, 519–540 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Milles, S. & Lemke, E.A. What precision-protein-tuning and nano-resolved single molecule sciences can do for each other. BioEssays 35, 65–74 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Los, G.V. & Wood, K. The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol. Biol. 356, 195–208 (2007).

    CAS  PubMed  Google Scholar 

  11. Miller, L.W., Cai, Y., Sheetz, M.P. & Cornish, V.W. In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag. Nat. Methods 2, 255–257 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl. Acad. Sci. USA 107, 10914–10919 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Uttamapinant, C. et al. Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angew. Chem. Int. Ed. Engl. 51, 5852–5856 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang, L. & Schultz, P.G. Expanding the genetic code. Angew. Chem. Int. Ed. Engl. 44, 34–66 (2004).

    Article  PubMed  Google Scholar 

  15. Liu, C.C. & Schultz, P.G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Lemke, E.A. The exploding genetic code. Chembiochem 15, 1691–1694 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Lang, K. & Chin, J.W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Wang, J., Xie, J. & Schultz, P.G. A genetically encoded fluorescent amino acid. J. Am. Chem. Soc. 128, 8738–8739 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Jewett, J.C. & Bertozzi, C.R. Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39, 1272–1279 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Agard, N.J., Prescher, J.A. & Bertozzi, C.R. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Wiessler, M., Waldeck, W., Kliem, C., Pipkorn, R. & Braun, K. The Diels-Alder-reaction with inverse-electron-demand, a very efficient versatile click-reaction concept for proper ligation of variable molecular partners. Int. J. Med. Sci. 7, 19–28 (2009).

    PubMed  PubMed Central  Google Scholar 

  22. Blackman, M.L., Royzen, M. & Fox, J.M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kolb, H.C., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. Engl. 40, 2004–2021 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective 'ligation' of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41, 2596–2599 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E.A. Genetically encoded copper-free click chemistry. Angew. Chem. Int. Ed. Engl. 50, 3878–3881 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Plass, T. et al. Amino acids for Diels-Alder reactions in living cells. Angew. Chem. Int. Ed. Engl. 51, 4166–4170 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Elliott, T.S. et al. Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat. Biotechnol. 32, 465–472 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nikic, I. et al. Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy. Angew. Chem. Int. Ed. Engl. 53, 2245–2249 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Raulf, A. et al. Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteins. RSC Adv. 4, 30462–30466 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Zhou, P. et al. Multicolor labeling of living-virus particles in live cells. Angew. Chem. Int. Ed. Engl. 51, 670–674 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Milles, S. et al. Click strategies for single-molecule protein fluorescence. J. Am. Chem. Soc. 134, 5187–5195 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Sun, S.B., Schultz, P.G. & Kim, C.H. Therapeutic applications of an expanded genetic code. Chembiochem 15, 1721–1729 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Patterson, D.M., Nazarova, L.A. & Prescher, J.A. Finding the right (bioorthogonal) chemistry. ACS Chem. Biol. 9, 592–605 (2014).

    Article  CAS  PubMed  Google Scholar 

  34. Liu, D.S. et al. Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. J. Am. Chem. Soc. 134, 792–795 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Uttamapinant, C., Sanchez, M.I., Liu, D.S., Yao, J.Z. & Ting, A.Y. Site-specific protein labeling using PRIME and chelation-assisted click chemistry. Nat. Protoc. 8, 1620–1634 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Lee, H.S., Guo, J., Lemke, E.A., Dimla, R.D. & Schultz, P.G. Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J. Am. Chem. Soc. 131, 12921–12923 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Summerer, D. et al. A genetically encoded fluorescent amino acid. Proc. Natl. Acad. Sci. USA 103, 9785–9789 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Herner, A. et al. New generation of bioorthogonally applicable fluorogenic dyes with visible excitations and large stokes shifts. Bioconjug. Chem. 25, 1370–1374 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Herner, A., Nikic, I., Kallay, M., Lemke, E.A. & Kele, P. A new family of bioorthogonally applicable fluorogenic labels. Org. Biomol. Chem. 11, 3297–3306 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Lukinavicius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Meimetis, L.G., Carlson, J.C., Giedt, R.J., Kohler, R.H. & Weissleder, R. Ultrafluorogenic coumarin-tetrazine probes for real-time biological imaging. Angew. Chem. Int. Ed. Engl. 53, 7531–7534 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions. J. Am. Soc. 134, 10317–10320 (2012).

    Article  CAS  Google Scholar 

  43. Borrmann, A. et al. Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation. Chembiochem 13, 2094–2099 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Parrish, A.R. et al. Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs. ACS Chem. Biol. 7, 1292–1302 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Greiss, S. & Chin, J.W. Expanding the genetic code of an animal. J. Am. Chem. Soc. 133, 14196–14199 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Carey, M.F., Peterson, C.L. & Smale, S.T. PCR-mediated site-directed mutagenesis. Cold Spring Harb. Protoc. 2013, 738–742 (2013).

    PubMed  Google Scholar 

  47. Pott, M., Schmidt, M.J. & Summerer, D. Evolved sequence contexts for highly efficient amber suppression with noncanonical amino acids. ACS Chem. Biol. 9, 2815–2822 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Beier, H. & Grimm, M. Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucleic Acids Res. 29, 4767–4782 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Phillips-Jones, M.K., Hill, L.S., Atkinson, J. & Martin, R. Context effects on misreading and suppression at UAG codons in human cells. Mol. Cell. Biol. 15, 6593–6600 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Yanagisawa, T. et al. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(ɛ)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem. Biol. 15, 1187–1197 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E. & Hayakawa, T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol. Ther. 1, 376–382 (2000).

    Article  CAS  PubMed  Google Scholar 

  52. Ryan, M.D. & Drew, J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13, 928–933 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 4, 298–304 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Sauer, J., Mielert, A., Lang, D. & Peter, D. Eine studie der Diels-Alder-reaktion, III: umsetzungen von 1.2.4.5-tetrazinen mit olefinen. Zur dtruktur von dihydropyridazinen. Chemische Berichte 98, 1435–1445 (1965).

    Article  CAS  Google Scholar 

  55. Kaya, E. et al. A genetically encoded norbornene amino acid for the mild and selective modification of proteins in a copper-free click reaction. Angew. Chem. Int. Ed. Engl. 51, 4466–4469 (2012).

    Article  CAS  PubMed  Google Scholar 

  56. Li, J., Jia, S. & Chen, P.R. Diels-Alder reaction-triggered bioorthogonal protein decaging in living cells. Nat. Chem. Biol. 10, 1003–1005 (2014).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the members of the Lemke and Schultz groups for proofreading and helpful discussions. We also thank SiChem for the gift of some of the compounds. This study was technically supported by the EMBL Advanced Light Microscopy Facility (ALMF) and the Proteomics Core Facility (PCF). I.N. acknowledges financial support by the European Commission Seventh Framework Programme (FP7) through Marie Curie Actions (FP7-PEOPLE-IEF) and a European Molecular Biology Organization (EMBO) long-term fellowship. E.A.L. acknowledges funding by the Emmy Noether program and SPP1623 of the Deutsche Forschungsgemeinschaft (DFG).

Author information

Author notes

  1. Ivana Nikić, Jun Hee Kang and Gemma Estrada Girona: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Ivana Nikić, Jun Hee Kang, Gemma Estrada Girona, Iker Valle Aramburu & Edward A Lemke

Authors
  1. Ivana Nikić
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun Hee Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gemma Estrada Girona
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Iker Valle Aramburu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Edward A Lemke
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

I.N., J.H.K. and G.E.G. performed the experiments and conceived and wrote the protocol; I.V.A. performed validation experiments; and E.A.L. conceived and wrote the protocol.

Corresponding author

Correspondence to Edward A Lemke.

Ethics declarations

Competing interests

Some of the compounds used in this work are covered by a patent application.

Integrated supplementary information

Supplementary Figure 1 Flow diagram of the protocol.

Supplementary Figure 2 Control strain-promoted alkyne-azide cycloaddition (SPAAC) labeling.

Unreactive non-canonical amino acid (BOC-Lys), IR(WT) or untransfected cells were labeled with Cy5-azide in the presence (a) and absence (b) of endocytosis blocker as done in main text Figure 3. Scale bar is 20μm.

Supplementary Figure 3 Strain-promoted alkyne-azide cycloaddition (SPAAC) short labeling.

Cells transfected with pIR(K676TAG)-GFP and expressing racBCN, endoBCN, exoBCN and SCO are labeled with 10 μM Cy5-azide dye for 10 min. Note that such labeling conditions (lower dye concentration and shorter incubation) did not lead to any noticeable labeling. Scale bar is 20μm.

Supplementary Figure 4 Control strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) labeling with H-Tet-Cy5.

Unreactive non-canonical amino acid (BOC-Lys), IR(WT) or untransfected cells with H-Tet-Cy5. Labeling conditions were as in main text figure 4. Note that no labeling is observed. Scale bar is 20μm.

Supplementary Figure 5 Control strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) labeling with Me-Tet-Cy5.

Unreactive non-canonical amino acid (BOC-Lys), IR(WT) or untransfected cells with Me-Tet-Cy5. Labeling conditions were as in main text figure 5. Scale bar is 20μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods (PDF 827 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nikić, I., Kang, J., Girona, G. et al. Labeling proteins on live mammalian cells using click chemistry. Nat Protoc 10, 780–791 (2015). https://doi.org/10.1038/nprot.2015.045

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.045

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

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