spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Poster Insert
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patterson, G.
Right arrow Articles by Piston, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patterson, G.
Right arrow Articles by Piston, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?
Journal of Cell Science 114, 837-838 (2001)
© 2001 The Company of Biologists Limited

CELL SCIENCE AT A GLANCE

Fluorescent protein spectra

George Patterson1, Rich N. Day2 and David Piston1,*

1 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, 37232-0615, USA
2 Departments of Medicine and Cell Biology, Box 800578 HSC, University of Virginia, Charlottesville, VA 22908-0578, USA

*Author for correspondence

The cloning of the green fluorescent protein (GFP) from the jellyfish Aequoria victoria and its expression in heterologous systems was a significant advance for optical microscopy of living cells (Chalfie et al., 1994). Mutagenesis of jellyfish GFP has yielded proteins that fluoresce from blue to yellowish green, and genetic manipulations have generated GFP variants that are better suited for fluorescence microscopy than wild-type GFP and have optimized codon usage (see Table 1; Tsien, R. Y., 1998). For example, a green color variant of Aequoria GFP (EGFP) has been extensively used as an in vivo reporter because of its high quantum yield and resistance to photobleaching. However, the useful cyan fluorescent variant (ECFP) has low absorption and low quantum yield, whereas the yellowish-green fluorescent protein (EYFP) has the highest absorption and quantum yield but is more susceptible to photobleaching than are most other mutants. The recent cloning of a gene that encodes a red fluorescent protein (dsRed) from the Indo-Pacific sea anemone Discosoma striata has provided yet another fluorescent protein that is further red-shifted (Matz, M. V. et al. 1999). The dsRed shares only ~25% sequence identity with Aequoria GFP; other usable GFPs are therefore likely to be discovered in the future. Several limitations to the use of dsRed have been identified, including slow protein maturation and a strong tendency to form tetramers (Baird, G. S., et al. 2000).


View this table:
[in this window]
[in a new window]
  		Table 1.
 
The poster shows excitation and emission spectra determined for each fluorescent protein (excitation spectra are shown in lighter shades). Methods for purification and characterization of fluorescent proteins are described in detail elsewhere (Piston et al. 1999). Briefly, the cDNA of each GFP was subcloned to produce an N-terminal His6 fusion protein. His6-tagged GFPs were expressed in E. coli grown at 37°C, and purified on a Ni NTA agarose column. Protein concentrations were determined by BCA assay, and the purification efficiencies were determined by scanning densitometry of SDS gels. Only proteins that were purified to >95% homogeneity were used for experiments. Extinction coefficients (see Table 1) were determined by Beers Law and data from an absorption spectrophotometer. Fluorescence excitation and emission spectra were measured and quantum yields were determined by using either fluorescein (QY = 0.85) or 1-aminoanthracene (QY = 0.61) as a reference standard. Photobleaching and pH stability measurements were performed as described previously (Patterson et al., 1997).

The fluorescent images shown in each panel are were obtained from the following proteins: GFP-Pit-1 (localized to the nucleus); YFP-STAT5B (localized throughout cell and concentrated in the nucleus); BFP-C/EBP{alpha} deletion 1-243 (localized to subnuclear foci); CFP-C/EBP{alpha} (localized to subnuclear foci); Ds-RED (throughout cell); GFP-GRIP-1 (subnuclear puncta), BFP-C/EBP{alpha} deletion 1-243 (subnuclear foci), and PML-DsRed (nuclear dots). In each case, cells were transfected with expression plasmids by electroporation, plated on glass coverslips and viewed after approximately 24 h in culture. The fluorescence images were acquired using an Olympus IX-70 inverted microscope (Olympus America, Melville, NY) equipped with a 60x aqueous-immersion objective lens and 100 W mercury-xenon arc lamp excitation light source. The detector used was a Hamamatsu Orca II cooled CCD camera.

Go



View larger version (37K):
[in this window]
[in a new window]
 
 

REFERENCES

Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Nat. Acad. Sci. USA 97, 11984-11989.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L. and Lukyanov, S. A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969-973.[Medline]

Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R. and Piston, D. W. (1997). Use of the green fluorescent protein (GFP) and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782-2790.[Medline]

Piston, D.W., G.H. Patterson, S.M. Knobel. (1999). Quantitative imaging of the green fluorescent protein (GFP). Meth. Cell Biol. 58, 31-48.[Medline]

Tsien, R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509-544.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:


Home page
 CSH ProtocolsHome page
M. A. Rizzo, M. W. Davidson, and D. W. Piston
Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants
CSH Protocols, December 1, 2009; 2009(12): pdb.top63 - pdb.top63.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
M. A. Rizzo, M. W. Davidson, and D. W. Piston
Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells
CSH Protocols, December 1, 2009; 2009(12): pdb.top64 - pdb.top64.
[Abstract] [Full Text]

Home page
 J. Cell Sci.Home page
M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown
Live-cell microscopy - tips and tools
J. Cell Sci., March 15, 2009; 122(6): 753 - 767.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
M. Lorenz
Visualizing protein-RNA interactions inside cells by fluorescence resonance energy transfer
RNA, January 1, 2009; 15(1): 97 - 103.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Chapman, C. Faulkner, E. Kaiserli, C. Garcia-Mata, E. I. Savenkov, A. G. Roberts, K. J. Oparka, and J. M. Christie
The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection
PNAS, December 16, 2008; 105(50): 20038 - 20043.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
V. Bayle, L. Nussaume, and R. A. Bhat
Combination of Novel Green Fluorescent Protein Mutant TSapphire and DsRed Variant mOrange to Set Up a Versatile in Planta FRET-FLIM Assay
Plant Physiology, September 1, 2008; 148(1): 51 - 60.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
T. H. Evers, M. A.M. Appelhof, E.W. Meijer, and M. Merkx
His-tags as Zn(II) binding motifs in a protein-based fluorescent sensor
Protein Eng. Des. Sel., August 1, 2008; 21(8): 529 - 536.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
V. L. Kolossov, B. Q. Spring, A. Sokolowski, J. E. Conour, R. M. Clegg, P. J. A. Kenis, and H. R. Gaskins
Engineering Redox-Sensitive Linkers for Genetically Encoded FRET-Based Biosensors
Experimental Biology and Medicine, February 1, 2008; 233(2): 238 - 248.
[Abstract] [Full Text] [PDF]

Home page
 JGPHome page
M. DiFranco, J. Capote, M. Quinonez, and J. L. Vergara
Voltage-dependent Dynamic FRET Signals from the Transverse Tubules in Mammalian Skeletal Muscle Fibers
J. Gen. Physiol., November 26, 2007; 130(6): 581 - 600.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. K. Gibson and A. G. Gilman
Gi{alpha} and G{beta} subunits both define selectivity of G protein activation by {alpha}2-adrenergic receptors
PNAS, January 3, 2006; 103(1): 212 - 217.
[Abstract] [Full Text] [PDF]

Home page
 MycologiaHome page
R. M. Andrie, J. P. Martinez, and L. M. Ciuffetti
Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes.
Mycologia, September 1, 2005; 97(5): 1152 - 1161.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
T. C. Voss, I. A. Demarco, C. F. Booker, and R. N. Day
Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus
J. Cell Sci., August 1, 2005; 118(15): 3277 - 3288.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
R. N. Day and F. Schaufele
Imaging Molecular Interactions in Living Cells
Mol. Endocrinol., July 1, 2005; 19(7): 1675 - 1686.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. E. Hatley, S. W. Lockless, S. K. Gibson, A. G. Gilman, and R. Ranganathan
Allosteric determinants in guanine nucleotide-binding proteins
PNAS, November 25, 2003; 100(24): 14445 - 14450.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Heck, O. Ermakova, H. Iwasaki, K. Akashi, C.-W. Sun, T. M. Ryan, T. Townes, and T. Graf
Distinguishable live erythroid and myeloid cells in beta -globin ECFP x lysozyme EGFP mice
Blood, February 1, 2003; 101(3): 903 - 906.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
M.-J. Gubbels, C. Li, and B. Striepen
High-Throughput Growth Assay for Toxoplasma gondii Using Yellow Fluorescent Protein
Antimicrob. Agents Chemother., January 1, 2003; 47(1): 309 - 316.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. A. Rizzo, M. A. Magnuson, P. F. Drain, and D. W. Piston
A Functional Link between Glucokinase Binding to Insulin Granules and Conformational Alterations in Response to Glucose and Insulin
J. Biol. Chem., September 6, 2002; 277(37): 34168 - 34175.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. V. Terskikh, A. F. Fradkov, A. G. Zaraisky, A. V. Kajava, and B. Angres
Analysis of DsRed Mutants. SPACE AROUND THE FLUOROPHORE ACCELERATES FLUORESCENCE DEVELOPMENT
J. Biol. Chem., March 1, 2002; 277(10): 7633 - 7636.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
G. Kang, O. G Chepurny, and G. G Holz
cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic {beta}-cells
J. Physiol., October 15, 2001; 536(2): 375 - 385.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor
Cell biology beyond the diffraction limit: near-field scanning optical microscopy
J. Cell Sci., January 12, 2001; 114(23): 4153 - 4160.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Poster Insert
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patterson, G.
Right arrow Articles by Piston, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patterson, G.
Right arrow Articles by Piston, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?