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:

Fluorescent labeling of tetracysteine-tagged proteins in intact cells

Nature Protocols volume 5, pages 1666–1677 (2010)Cite this article

Subjects

Abstract

In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed.

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: Time dependence of FlAsH binding to the target sequence.
Figure 2: Specific labeling with FlAsH can only be seen after appropriate washing.
Figure 3: Reduction of FlAsH binding to different tetracysteine motifs by BAL.
Figure 4: Comparison of BAL and EDT for removing FlAsH from its target sequences.
Figure 5: Labeling of the cytosolic protein β-arrestin-2 with FlAsH.
Figure 6: Dynamic FRET measurements between CFP and FlAsH.
Figure 7: Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes.

Similar content being viewed by others

References

  1. Sletten, E.M. & Bertozzi, C.R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. Engl. 48, 6974–6998 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. 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 

  5. Gronemeyer, T., Godin, G. & Johnsson, K. Adding value to fusion proteins through covalent labelling. Curr. Opin. Biotechnol. 16, 453–458 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. & Johnsson, K. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl. Acad. Sci. USA 101, 9955–9959 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Maurel, D. et al. Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat. Methods 5, 561–567 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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 

  9. Los, G.V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Soh, N. Selective chemical labeling of proteins with small fluorescent molecules based on metal-chelation methodology. Sensors 8, 1004–1102 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Hauser, C.T. & Tsien, R.Y. A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure. Proc. Natl. Acad. Sci. USA 104, 3693–3697 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Kapanidis, A.N., Ebright, Y.W. & Ebright, R.H. Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with Ni2+:nitrilotriacetic acid (n)-fluorochrome conjugates. J. Am. Chem. Soc. 123, 12123–12125 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Guignet, E.G., Hovius, R. & Vogel, H. Reversible site-selective labeling of membrane proteins in live cells. Nat. Biotechnol. 22, 440–444 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Litowski, J.R. & Hodges, R.S. Designing heterodimeric two-stranded alpha-helical coiled-coils. Effects of hydrophobicity and alpha-helical propensity on protein folding, stability, and specificity. J. Biol. Chem. 277, 37272–37279 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Yano, Y. et al. Coiled-coil tag-probe system for quick labeling of membrane receptors in living cell. ACS Chem. Biol. 3, 341–345 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. 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 

  18. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Stroffekova, K., Proenza, C. & Beam, K.G. The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins. Pflügers Arch. 442, 859–866 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. 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 

  21. Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Wang, T., Yan, P., Squier, T.C. & Mayer, M.U. Prospecting the proteome: identification of naturally occurring binding motifs for biarsenical probes. Chembiochem 8, 1937–1940 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Spagnuolo, C.C., Vermeij, R.J. & Jares-Erijman, E.A. Improved photostable FRET-competent biarsenical-tetracysteine probes based on fluorinated fluoresceins. J. Am. Chem. Soc. 128, 12040–12041 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Adams, S.R. & Tsien, R.Y. Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins. Nat. Protoc. 3, 1527–1534 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Madani, F. et al. Hairpin structure of a biarsenical-tetracysteine motif determined by NMR spectroscopy. J. Am. Chem. Soc. 131, 4613–4615 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Martin, B.R., Deerinck, T.J., Ellisman, M.H., Taylor, S.S. & Tsien, R.Y. Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells. Chem. Biol. 14, 1031–1042 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Chaumont, S. & Khakh, B.S. Patch-clamp coordinated spectroscopy shows P2X2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels. Proc. Natl. Acad. Sci. USA 105, 12063–12068 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Hoffmann, C. et al. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat. Methods 2, 171–176 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Nikolaev, V.O., Hoffmann, C., Bünemann, M., Lohse, M.J. & Vilardaga, J.P. Molecular basis of partial agonism at the neurotransmitter a2A-adrenergic receptor and Gi-protein heterotrimer. J. Biol. Chem. 281, 24506–24511 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Böhme, I., Morl, K., Bamming, D., Meyer, C. & Beck-Sickinger, A.G. Tracking of human Y receptors in living cells—a fluorescence approach. Peptides 28, 226–234 (2007).

    Article  PubMed  Google Scholar 

  31. Ju, W. et al. Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors. Nat. Neurosci. 7, 244–253 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Enninga, J., Mounier, J., Sansonetti, P. & Tran Van Nhieu, G. Secretion of type III effectors into host cells in real time. Nat. Methods 2, 959–965 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Ignatova, Z. & Gierasch, L.M. Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc. Natl. Acad. Sci. USA 101, 523–528 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Rudner, L. et al. Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling. J. Virol. 79, 4055–4065 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Das, S.C., Panda, D., Nayak, D. & Pattnaik, A.K. Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells. J. Virol. 83, 2611–2622 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Venken, K.J. et al. Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation. Nucleic Acids Res. 36, e114 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R. & Tsien, R.Y. Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Marek, K.W. & Davis, G.W. Transgenically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I. Neuron 36, 805–813 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Roberti, M.J., Bertoncini, C.W., Klement, R., Jares-Erijman, E.A. & Jovin, T.M. Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein. Nat. Methods 4, 345–351 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Chen, B., Mayer, M.U., Markillie, L.M., Stenoien, D.L. & Squier, T.C. Dynamic motion of helix A in the amino-terminal domain of calmodulin is stabilized upon calcium activation. Biochemistry 44, 905–914 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Jost, C.A., Reither, G., Hoffmann, C. & Schultz, C. Contribution of fluorophores to protein kinase C FRET probe performance. Chembiochem 9, 1379–1384 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Zürn, A. et al. Fluorescence resonance energy transfer analysis of a2A-adrenergic receptor activation reveals distinct agonist-specific conformational changes. Mol. Pharmacol. 75, 534–541 (2009).

    Article  PubMed  Google Scholar 

  43. Luedtke, N.W., Dexter, R.J., Fried, D.B. & Schepartz, A. Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH. Nat. Chem. Biol. 3, 779–784 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Chen, B., Cao, H., Yan, P., Mayer, M.U. & Squier, T.C. Identification of an orthogonal peptide binding motif for biarsenical multiuse affinity probes. Bioconjug. Chem. 18, 1259–1265 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Zürn, A. et al. Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores. Bioconjug. Chem. 21, 853–859 (2010).

    Article  PubMed  Google Scholar 

  46. Van Engelenburg, S.B., Nahreini, T. & Palmer, A.E. FACS-based selection of tandem tetracysteine peptides with improved ReAsH brightness in live cells. Chembiochem 11, 489–493 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Andresen, M., Schmitz-Salue, R. & Jakobs, S. Short tetracysteine tags to beta-tubulin demonstrate the significance of small labels for live cell imaging. Mol. Biol. Cell 15, 5616–5622 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Langhorst, M.F., Genisyuerek, S. & Stuermer, C.A. Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by b-mercaptoethanol for specific staining of tetracysteine-tagged proteins. Histochem Cell Biol. 125, 743–747 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Tour, O. et al. Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator. Nat. Chem. Biol. 3, 423–431 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Gaietta, G.M. et al. Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy. Proc. Natl. Acad. Sci. USA 103, 17777–17782 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Griffin, B.A., Adams, S.R., Jones, J. & Tsien, R.Y. Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol. 327, 565–578 (2000).

    Article  CAS  PubMed  Google Scholar 

  52. Campbell, R.E. Fluorescent-protein-based biosensors: modulation of energy transfer as a design principle. Anal. Chem. 81, 5972–5979 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Maier-Peuschel, M. et al. A fluorescence resonance energy transfer-based M2 muscarinic receptor sensor reveals rapid kinetics of allosteric modulation. J. Biol. Chem. 285, 8793–8800 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Robia, S.L., Flohr, N.C. & Thomas, D.D. Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy. Biochemistry 44, 4302–4311 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Research in the authors' laboratories is supported by the US National Institutes of Health, the Howard Hughes Medical Institute, the Deutsche Forschungsgemeinschaft (German Research Foundation) and the European Research Council.

Author information

Author notes

  1. Sonia Terrillon

    Present address: Present address: Department of Molecular & Cellular Biology, Abbott Bioresearch Center, Worcester, Massachusetts, USA.,

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

    Carsten Hoffmann, Alexander Zürn & Martin J Lohse

  2. Department of Neurosciences, National Center of Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, USA

    Guido Gaietta & Mark H Ellisman

  3. Department of Pharmacology, University of California, San Diego, La Jolla, California, USA

    Stephen R Adams, Sonia Terrillon & Roger Y Tsien

Authors
  1. Carsten Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guido Gaietta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander Zürn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen R Adams
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sonia Terrillon
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mark H Ellisman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Roger Y Tsien
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Martin J Lohse
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.H., G.G., A.Z., S.R.A. and S.T. conducted experiments; C.H. and G.G. drafted the paper; and M.H.E., R.Y.T. and M.J.L. finalized the paper.

Corresponding author

Correspondence to Martin J Lohse.

Ethics declarations

Competing interests

The University of California, San Diego owns a patent on FlAsH, ReAsH, and tetracysteine sequences. R.Y.T. shares inventors' royalties from these patents. The University of Washington owns a patent on fluorescent GPCR sensors. C.H. and M.J.L. share inventors' royalties from these patents.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hoffmann, C., Gaietta, G., Zürn, A. et al. Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat Protoc 5, 1666–1677 (2010). https://doi.org/10.1038/nprot.2010.129

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.129

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