Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo

Jason A. Kahana1, Pam A. Silver1

1 Dana‐Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.6
DOI:  10.1002/0471141755.ph0606s05
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

PDF or HTML at Wiley Online Library

Table of Contents

  • Overview of GFP Fluorescence
  • Utilization of GFP
  • Problems with GFP
  • Mutants of GFP
  • Microscopy Setup
  • Conclusions
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802‐805.
   Cody, C.W., Prasher, D.C., Westler, W.M., Pendergast, F.G., and Ward, W.W. 1993. Chemical structure of the hexapeptide chromophore of the Aequorea green‐fluorescent protein. Biochemistry 32:1212‐1218.
   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. Bio/Technology 13:151‐154.
   Flach, J., Bossie, M., Vogel, J., Corbett, A.H., Jinks, T., Willins, D.A., and Silver, P.A. 1994. A yeast RNA‐binding protein shuttles between the nucleus and the cytoplasm. Mol. Cell. Biol. 14:8399‐8407.
   Haseloff, J. and Amos, B. 1995. GFP in plants. Trends Genet. 11:328‐329.
   Hecht, E. 1987. Optics, 2nd ed. Addison‐Wesley Publishing, Reading, Mass.
   Heim, R., Cubitt, A.B., and Tsien, R.Y. 1995. Improved green fluorescence. Nature 373:663‐664.
   Heim, R., Prasher, D.C., and Tsien, R.Y. 1994. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 91:12501‐12504.
   Inouye, S. and Tsuji, F.I. 1994a. Aequorea green fluorescent protein: Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett. 341:277‐280.
   Inouye, S. and Tsuji, F.I. 1994b. Evidence for redox forms of the Aequorea green fluorescent protein. FEBS Lett. 351:211‐214.
   Kahana, J.A., Schnapp, B.J., and Silver, P.A. 1995. Kinetics of spindle pole body separation in budding yeast. Proc. Natl. Acad. Sci. U.S.A. 92:9707‐9711.
   Morin, J.G. and Hastings, J.W. 1971. Energy transfer in a bioluminescent system. J. Cell Physiol. 77:313‐318.
   Morise, H., Shimomura, O., Johnson, F.H., and Winant, J. 1974. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13:2656‐2663.
   Olson, K.R., McIntosh, J.R., and Olmstead, J.B. 1995. Analysis of MAP4 function in living cells using green fluorescent protein (GFP) chimeras. J. Cell Biol. 130:639‐650.
   Pines, J. 1995. GFP in mammalian cells. Trends Genet. 11:326‐327.
   Prasher, D.C., Eckenrode, V.K., Ward, W.W., Pendergast, F.G., and Cormier, M.J. 1992. Primary structure of the Aequorea victoria green‐fluorescent protein. Gene 111:229‐233.
   Reichman, J. 1994. Handbook of Optical Filters for Fluorescence Microscopy. Chroma Technology Corporation, Brattleboro, Vt.
   Wang, S. and Hazelrigg, T. 1994. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369:400‐402.
   Ward, W.W. 1981. Properties of the coelenterate green fluorescent proteins. In Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications (M.A. DeLuca and W.D. McElroy, eds.) pp. 225‐234. Academic Press, San Diego.
   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.
   Ward, W.W., Cody, C.W., Hart, R.C., and Cormier, M.J. 1980. Spectrophotometric identity of the energy transfer chromophores in Renilla and Aequorea green‐fluorescent proteins. Photochem. Photobiol. 31:611‐615.
   Yocum, R.R., Hanley, S., West, R., and Ptashne, M. 1984. Use of LacZ fusions to delimit regulatory elements of the inducible divergent GAL1‐GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:1985‐1998.
PDF or HTML at Wiley Online Library