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Cell-Surface Protein–Protein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies

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Cell-Cell Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1066))

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Abstract

Förster resonance energy transfer (FRET) is a proximity-dependent quantum effect that allows the measurement of protein interactions and conformational changes which are invisible to traditional forms of fluorescence or electron microscopy. However, FRET experiments often have difficulty detecting interactions that are transient and localized or occur in low abundance against a large background. This protocol describes a method of improving on the sensitivity and quantifiability of FRET experiments by using time-specific detection to isolate FRET-mediated acceptor emission from cross-talk excitation and all other sources of nonspecific fluorescence background.

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References

  1. Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7:730–734

    Article  PubMed  CAS  Google Scholar 

  2. Gould TJ, Hess ST, Bewersdorf J (2012) Optical nanoscopy: from acquisition to analysis. Annu Rev Biomed Eng 14:231–254

    Article  PubMed  CAS  Google Scholar 

  3. Lam AJ, St-Pierre F, Gong Y et al (2012) Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9:1005–1012

    Article  PubMed  CAS  Google Scholar 

  4. Xia Z, Liu Y (2001) Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J 81:2395–2402

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  6. Bazin H, Trinquet E, Mathis G (2002) Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. J Biotechnol 82:233–250

    PubMed  CAS  Google Scholar 

  7. Albizu L, Cottet M, Kralikova M et al (2010) Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol 6:587–594

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  9. Yano Y, Matsuzaki K (2009) Tag-probe labeling methods for live-cell imaging of membrane proteins. Biochim Biophys Acta 1788: 2124–2131

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  11. Huang Y, Willars GB (2011) Generation of epitope-tagged GPCRs. Methods Mol Biol 746:53–84

    Article  PubMed  CAS  Google Scholar 

  12. Braman J, Papworth C, Greener A (1996) Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol 57:31–44

    PubMed  CAS  Google Scholar 

  13. Bylund DB, Deupree JD, Toews ML (2004) Radioligand-binding methods for membrane preparations and intact cells. Methods Mol Biol 259:1–28

    PubMed  CAS  Google Scholar 

  14. Hoffmann C, Gaietta G, Bunemann M et al (2005) A FlAsH-based FRET approach to determine G protein-coupled receptors: the quest for functionally selective conformations is open. Nat Methods 2:171–176

    Article  PubMed  CAS  Google Scholar 

  15. Petralia RS, Wenthold RJ (1999) Immunocytochemistry of NMDA receptors. Methods Mol Biol 128:73–92

    PubMed  CAS  Google Scholar 

  16. Lakshmipathy U, Buckley S, Verfaillie C (2007) Gene transfer Via nucleofectin into adult and embryonic stem cells. Methods Mol Biol 407:115–126

    Article  PubMed  CAS  Google Scholar 

  17. Gaastra W (1984) Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol 1:349–355

    PubMed  CAS  Google Scholar 

  18. Tramier M, Piolot T, Gautier I et al (2003) Homo-FRET versus hetero-FRET to probe homodimers in living cells. Methods Enzymol 360:580–597

    Article  PubMed  CAS  Google Scholar 

  19. Mercier JF, Salahpour A, Angers S et al (2002) Quantitative assessment of ®1- and ®2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277:44925–44931

    Article  PubMed  CAS  Google Scholar 

  20. Zacharias DA, Violin JD, Newton AC et al (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916

    Article  PubMed  CAS  Google Scholar 

  21. Herman B, Krishnan RV, Centonze VE (2004) Microscopic analysis of fluorescence resonance energy transfer (FRET). Methods Mol Biol 261:351–370

    PubMed  CAS  Google Scholar 

  22. Ward RJ, Pediani JD, Milligan G (2011) Ligand-induced internalization of the orexin OX(1) and cannabinoid CB(1) receptors assessed via N-terminal SNAP and CLIP-tagging. Br J Pharmacol 162:1439–1452

    Article  PubMed  CAS  Google Scholar 

  23. Vilardaga JP, Nikolaev VO, Lorenz K et al (2008) Direct inhibition of G protein signaling by cross-conformational switches between a2A-adrenergic and μ-opioid receptors. Nat Chem Biol 4:126–131

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    Timothy N. Feinstein

Authors
  1. Timothy N. Feinstein
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Editor information

Editors and Affiliations

  1. Texas A&M Health Science Center, Temple, Texas, USA

    Troy A. Baudino

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© 2013 Springer Science+Business Media, New York

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Feinstein, T.N. (2013). Cell-Surface Protein–Protein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies. In: Baudino, T. (eds) Cell-Cell Interactions. Methods in Molecular Biology, vol 1066. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-604-7_11

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  • DOI: https://doi.org/10.1007/978-1-62703-604-7_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-603-0

  • Online ISBN: 978-1-62703-604-7

  • eBook Packages: Springer Protocols

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