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BiFC for Protein–Protein Interactions and Protein Topology: Discussing an Integrative Approach for an Old Technique

  • Giovanni Stefano
  • Luciana Renna
  • Federica BrandizziEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1242)

Abstract

BiFC (Bimolecular Fluorescence Complementation) is one of the most widely used techniques to study protein–protein interactions as well as protein topology in living cells. This method allows the visualization of protein interactions or the analysis of their topology in the cell compartments where the proteins belong, without changing their chemical properties, as often occurs after mixing the content of different cellular compartments in cell extracts. Several laboratories use this method because it is relatively easy to perform; however, sometimes a positive protein–protein interaction BiFC signal (i.e., reconstitution of fluorescence of interacting protein pairs) does not necessarily mean that the tested proteins are actually interacting in vivo in a specific way. Here we describe the BiFC approach for assessing protein–protein interactions and for establishing protein topology and we discuss how to best perform this method to avoid false positive results when studying protein interactions in plant cells.

Key words

Bimolecular fluorescence complementation BiFC Tobacco Protein–protein interactions YFP Fluorescent protein 

Notes

Acknowledgements

We acknowledge support by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (award number DE-FG02-91ER20021) for infrastructure support and National Science Foundation (MCB 1243792).

References

  1. 1.
    Hu CD, Chinenov Y, Kerppola TK (2002) Mol Cell 9:789–798PubMedCrossRefGoogle Scholar
  2. 2.
    Citovsky V, Lee LY, Vyas S, Glick E, Chen MH, Vainstein A, Gafni Y, Gelvin SB, Tzfira T (2006) J Mol Biol 362:1120–1131PubMedCrossRefGoogle Scholar
  3. 3.
    Schutze K, Harter K, Chaban C (2009) Methods Mol Biol 479:189–202PubMedCrossRefGoogle Scholar
  4. 4.
    Hu CD, Kerppola TK (2003) Nat Biotechnol 21:539–545PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Mehrshahi P, Stefano G, Andaloro JM, Brandizzi F, Froehlich JE, Dellapenna D (2013) Proc Natl Acad Sci U S A 110:12126–12131PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Zamyatnin AA Jr, Solovyev AG, Bozhkov PV, Valkonen JP, Morozov SY, Savenkov EI (2006) Plant J 46:145–154PubMedCrossRefGoogle Scholar
  7. 7.
    Sparkes I, Brandizzi F (2012) Plant J 70:96–107PubMedCrossRefGoogle Scholar
  8. 8.
    Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Gene 77:51–59PubMedCrossRefGoogle Scholar
  9. 9.
    Higuchi R, Krummel B, Saiki RK (1988) Nucleic Acids Res 16:7351–7367PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Slabaugh E, Held M, Brandizzi F (2011) Plant J 67:395–405PubMedCrossRefGoogle Scholar
  11. 11.
    Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Plant J 40:428–438PubMedCrossRefGoogle Scholar
  12. 12.
    Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) Plant J 64:355–365PubMedCrossRefGoogle Scholar
  13. 13.
    Sparkes IA, Runions J, Kearns A, Hawes C (2006) Nat Protoc 1:2019–2025PubMedCrossRefGoogle Scholar
  14. 14.
    Kerppola TK (2013) Cold Spring Harb Protoc 2013:727–731PubMedGoogle Scholar
  15. 15.
    Kerppola TK (2013) Cold Spring Harb Protoc 2013:714–718PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Giovanni Stefano
    • 1
  • Luciana Renna
    • 1
  • Federica Brandizzi
    • 1
    Email author
  1. 1.MSU-DOE Plant Research LabMichigan State UniversityEast LansingUSA

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