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Fluorescence Gel Retardation Assay to Detect Protein-Protein Interactions

  • Sang-Hyun Park
  • Ronald T. Raines
Part of the Methods in Molecular Biology book series (MIMB, volume 261)

Abstract

A gel mobility retardation assay can be used to detect a protein-protein interaction. The assay is based on the electrophoretic mobility of a protein-protein complex being less than that of either protein alone. Electrophoretic mobility is detected by the fluorescence of a green fluorescent protein variant that is fused to one of the protein partners. The assay is demonstrated by using the interaction of the S-protein and S-peptide fragments of ribonuclease A as a case study.

Key Words

Electrophoresis fusion protein gel mobility shift gel retardation green fluorescent protein protein-protein interaction 

References

  1. 1.
    Carey, J. (1991) Gel retardation. Methods Enzymol. 208, 103–117.PubMedCrossRefGoogle Scholar
  2. 2.
    Park, S.-H. and Raines, R. T. (1997) Green fluorescent protein as a signal for protein-protein interactions. Protein Sci. 6, 2344–2349.PubMedCrossRefGoogle Scholar
  3. 3.
    Park, S. H. and Raines, R. T. (2000) Green fluorescent protein chimeras to probe protein-protein interactions. Methods Enzymol. 328, 251–261.PubMedCrossRefGoogle Scholar
  4. 4.
    Bokman, S. H. and Ward, W. W. (1981) Renaturation of Aequorea green fluorescent protein. Biochem. Biophys. Res. Commun. 101, 1372–1380.PubMedCrossRefGoogle Scholar
  5. 5.
    Ward, W. W. (1981) in Bioluminescence and Chemiluminescence (DeLuca, M., and McElroy, W., eds), Academic Press, New York, NY, pp. 235–242.Google Scholar
  6. 6.
    Ward, W. W. and Bokman, S. H. (1982) Reversible denaturation of Aequorea green fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21, 4535–4540.PubMedCrossRefGoogle Scholar
  7. 7.
    Hirschberg, K., Phair, R. D., and Lippincott-Schwartz, J. (2000) Kinetic analysis of intracellular trafficking in single living cells with vesicular stomatitis virus protein G-green fluorescent protein hybrids. Methods Enzymol. 327, 69–89.Google Scholar
  8. 8.
    Meyer, T. and Oancea, E. (2000) Studies of signal transduction events using chimeras to green fluorescent protein. Methods Enzymol. 327, 500–513.PubMedCrossRefGoogle Scholar
  9. 9.
    Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aeqourea victoria green-fluorescent protein. Gene 111, 229–233.PubMedCrossRefGoogle Scholar
  10. 10.
    Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455.PubMedCrossRefGoogle Scholar
  11. 11.
    Delagrave, S., Hawtin, R. E., Silva, C. M., Yang, M. M., and Youvan, D. C. (1995) Red-shifted excitation mutants of the green fluorescent protein. BioTechnology 13, 151–154.PubMedCrossRefGoogle Scholar
  12. 12.
    Ehrig, T., O’Kane, D. J., and Prendergast, F. G. (1995) Green-fluorescent protein with altered fluorescence excitation spectra. FEBS Lett. 367, 163–166.PubMedCrossRefGoogle Scholar
  13. 13.
    Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.PubMedCrossRefGoogle Scholar
  14. 14.
    Crameri, A., Whitehorn, E. A., Tate, E., and Stemmer, W. P. C. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14, 315–319.CrossRefGoogle Scholar
  15. 15.
    Ward, W. W. (1997) Green Fluorescent Protein: Properties, Applications and Protocols (Chalfie, M., and Kain, S., eds.), Wiley, New York, NY.Google Scholar
  16. 16.
    Ormö, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., and Remington, S. J. (1996) Crystal structure of the Aeqourea victoria green fluorescent protein. Science 237, 1392–1395.CrossRefGoogle Scholar
  17. 17.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  18. 18.
    Forwood, J. K., Lam, M. H., and Jans, D. A. (2001) Nuclear import of Creb and AP-1 transcription factors requires importin-β1 and Ran but is independent of importin-α. Biochemistry 40, 5208–5217.PubMedCrossRefGoogle Scholar
  19. 19.
    Kiessig, S., Reissmann, J., Rascher, C., Kullertz, G., Fischer, A., and Thunecke, F. (2001) Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods. Electrophoresis 22, 1428–1435.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Sang-Hyun Park
    • 1
  • Ronald T. Raines
    • 2
  1. 1.Department of Cellular and Molecular PharmacologyUniversity of CaliforniaSan Francisco
  2. 2.Departments of Biochemistry and ChemistryUniversity of Wisconsin-MadisonMadison

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