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Intensive searches for novel green fluorescent protein (GFP)-like fluorescent proteins have identified more than 150 distinct genes that, together with its mutants, cover the excitation range from 380 to 600 nanometers (nm) and the emission range from 440 to 650 nm (see table below). Despite spectral diversity, a family of GFP-like proteins possesses common significant structural, biochemical and photophysical features. Many of these spectroscopically active proteins are developed to commercially available genetically-encoded fluorescent probes. In comparison to other natural pigments and fluorophores, GFP-like proteins stand out because they form internal chromophores without requiring accessory cofactors, external enzymatic catalysis or substrates other than molecular oxygen. It gives GFP-like proteins many advantages including that chromophore formation is possible in live organisms, tissues or cells while maintaining their integrity as well as molecular, organelle and tissue targeting and specificity.

Fluorescent proteins can be divided into several fluorescent groups with respect to the appearance of the purified protein to the human eye:

  • Blue (below 460 nm, BFP)
  • Cyan (460-500 nm, CFP)
  • Green (~500-520 nm, GFP)
  • Yellow (~520-550 nm, YFP)
  • Orange (~550-570 nm, OFP)
  • Red (~570-620 nm, RFP)
  • Far red (above 620 nm, FRFP)

In addition, several fluorescent proteins exhibit photoactivatable (PA-FP) or photoswitchable behavior and therefore are called photoactivatable (PA-FP) or photoswitchable (PS-FP) fluorescent proteins, respectively. These proteins are originally either dark (PA-FP) or fluoresce at one wavelength (PS-FP) but become fluorescent or fluorescent at a distinct wavelength, respectively, upon irradiation with an intense violet or blue light. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness. Flow cytometers currently available at the core facility allow for simultaneous detection of many fluorescent proteins of different fluorescent groups simultaneously expressed in the cells.

The following list is not exhaustive. It illustrates the properties of recommended fluorescent proteins that were available at the time that the table was last updated, which happens regularly.

Properties of Recommended Fluorescent Proteins

Protein NamesReference or SourceSpectral PropertiesOligomeric StateAECOM core facility flow cytometer
Peak Excitation
Peak Emission
Brightness (relative to eGFP)FACScanLRSIIMoFloMoFlo XDPFACSAriaForcheimer FACSAria
Blue Fluorescent Proteins
EBFPYang et al.m J. Biol. Chem., 1998, 273, 82123804400.27monomer 
AzuriteMena et al., Nat. Biotechnol., 2006, 24, 15693834470.43monomer 
EBFP2Ai et al., Biochemistry, 2007, 46, 59043834480.60monomer 
SuperGlo BFPwww.qbiogene.com387450n/amonomer 
Cyan Fluorescent Proteins
CeruleanRizzo et al., Nat. Biotechnol., 2004, 22, 4454334750.79monomer 
CyPetNguyen et al., Nat. Biotechnol., 2005, 23, 3554354770.53monomer 
TagCFPwww.evrogen.com4584800.84dimer √ *√ *√ *
mTFP1Ai et al., Biochem. J., 2006, 400, 5314624921.58dimer √ *√ *√ *
Midoriishi Cyanwww.mblintl.com4724950.73dimer √ *√ *√ *
Green Fluorescent Proteins
Azami Greenwww.mblintl.com4925051.21monomer
EmeraldCubitt et al., Methods Cell. Biol., 1999, 58, 194875091.16monomer
T-SapphireZapata-Hommer et al., BMC Biotechnol., 2003, 3, 53995110.78monomer 
Yellow Fluorescent Proteins
TopazCubitt et al., Methods Cell. Biol., 1999, 58, 195145271.69monomer
VenusNagai et al., Nat. Biotechnol., 2002, 20, 875155281.56monomer
mCitrineGriesbeck et al., J. Biol. Chem., 2001, 276, 291885165291.74monomer
YPetNguyen et al., Nat. Biotechnol., 2005, 23, 3555175302.38monomer
Orange Fluorescent Proteins
Kusabira Orangewww.mblintl.com5485590.92monomer   
mOrangeShaner et al., Nat. Biotechnol., 2004, 22, 15245485621.46monomer   
Red Fluorescent Proteins
dTomatoShaner et al., Nat. Biotechnol., 2004, 22, 15245545811.42dimer  
Express (T1)
TagRFPMerzlyak et al., Nat. Methods, 2007, 4, 555
mStrawberryShaner et al., Nat. Biotechnol., 2004, 22, 15245745960.78monomer  
mCherryShaner et al., Nat. Biotechnol., 2004, 22, 15245876100.47monomer  
mKeimaKogure et al., Nat. Biotechnol., 2006, 24, 5774406200.12monomer √ *√ *√ *√ *√ *
Far-Red Fluorescent Proteins
mRaspberryWang et al., PNAS, 2004, 101, 167455986250.37monomer     
KatushkaShcherbo et al., Nat. Methods, 2007, 4, 7415886350.67dimer     
Shcherbo et al., Nat. Methods, 2007, 4, 741
HcRedGurskaya et al., FEBS Lett., 2001, 507, 165926450.05tetramer     
mPlumWang et al., PNAS, 2004, 101, 167455906490.12monomer     
AQ143Shkrob et al., Biochem. J., 2005, 392, 6495956550.11tetramer     
PA-GFP     Patterson et al., Science, 2002, 297, 1873
before activation     4005150.08monomer 
after activation     5045170.42 
PS-CFP2     www.evrogen.com
before activation     4004700.26monomer 
after activation     4905110.33 
Dronpa     www.mblintl.com
before activation     n/an/a<0.01monomer
after activation     5035182.45
Kaede     www.mblintl.com
before activation     5085182.64tetramer 
after activation     5725800.60  
mEosFP     Weidemann et al., PNAS, 2004, 101, 15905
before activation     5055161.30monomer
after activation     5695810.70  
Dendra2     www.evrogen.com
before activation     4905070.45monomer
after activation     5535730.39  
KFP-Red     www.evrogen.com
before activation     n/an/a<0.01tetramern/an/an/an/an/an/a
after activation     5806000.13  
  * Excitation is suboptimal using the cytometer's existing laser lines.

Recommended Reviews on fluorescent Proteins:
1.   Verkhusha V.V. et al. The molecular properties and applications of Anthozoa fluorescent proteins and chromophores. Nature Biotechnology. 2004, 22, 289-296.
2.   Shaner N.C. et al. Perspective: A guide to choosing fluorescent proteins. Nature Methods. 2005, 2, 905-909.
3.   Chudakov D.M. et al. Fluorescent proteins as a toolkit for in vivo imaging. Trends in Biotechnology. 2005, 23, 605-613.
4.   Lukyanov K.A. et al. Innovation: Photoactivatable fluorescent proteins. Nature Rev. Mol. Cell Biol. 2005, 6, 885-891.
5.   Giepmans B.N.G. et al. The fluorescent toolbox for assessing protein location and function. Science, 2006, 312, 217-224.
6.   Smith C. Keeping tabs on fluorescent tags. Nature Methods. 2007, 4, 755-761.
Internet resources and commercially available fluorescent proteins:
1.   Olympus
2.   Clontech Laboratories
3.   Evrogen
4.   Invitrogen Molecular Probes
5.   NanoLight Technology
6.   Florida State University
With further questions on fluorescent proteins contact Dr. Vlad Verkhusha at the Department of Anatomy and Structural Biology.

Jinghang Zhang is solely responsible for the content of this site. Comments, concerns and questions regarding it should be addressed to her.
File Last Updated: Monday, January 12, 2009