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.
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References
Carey, J. (1991) Gel retardation. Methods Enzymol. 208, 103–117.
Park, S.-H. and Raines, R. T. (1997) Green fluorescent protein as a signal for protein-protein interactions. Protein Sci. 6, 2344–2349.
Park, S. H. and Raines, R. T. (2000) Green fluorescent protein chimeras to probe protein-protein interactions. Methods Enzymol. 328, 251–261.
Bokman, S. H. and Ward, W. W. (1981) Renaturation of Aequorea green fluorescent protein. Biochem. Biophys. Res. Commun. 101, 1372–1380.
Ward, W. W. (1981) in Bioluminescence and Chemiluminescence (DeLuca, M., and McElroy, W., eds), Academic Press, New York, NY, pp. 235–242.
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.
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.
Meyer, T. and Oancea, E. (2000) Studies of signal transduction events using chimeras to green fluorescent protein. Methods Enzymol. 327, 500–513.
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.
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.
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.
Ehrig, T., O’Kane, D. J., and Prendergast, F. G. (1995) Green-fluorescent protein with altered fluorescence excitation spectra. FEBS Lett. 367, 163–166.
Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.
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.
Ward, W. W. (1997) Green Fluorescent Protein: Properties, Applications and Protocols (Chalfie, M., and Kain, S., eds.), Wiley, New York, NY.
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.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
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.
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.
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© 2004 Humana Press Inc., Totowa, NJ
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Park, SH., Raines, R.T. (2004). Fluorescence Gel Retardation Assay to Detect Protein-Protein Interactions. In: Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 261. Humana Press. https://doi.org/10.1385/1-59259-762-9:155
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DOI: https://doi.org/10.1385/1-59259-762-9:155
Publisher Name: Humana Press
Print ISBN: 978-1-58829-120-2
Online ISBN: 978-1-59259-762-8
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