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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 Names	Reference or Source	Spectral Properties			Oligomeric State	AECOM core facility flow cytometer
Protein Names	Reference or Source	Peak Excitation nm	Peak Emission nm	Brightness (relative to eGFP)	Oligomeric State	FACScan	LRSII	MoFlo	MoFlo XDP	FACSAria	Forcheimer FACSAria
Blue Fluorescent Proteins
EBFP	Yang et al.m J. Biol. Chem., 1998, 273, 8212	380	440	0.27	monomer		√	√	√	√	√
Azurite	Mena et al., Nat. Biotechnol., 2006, 24, 1569	383	447	0.43	monomer		√	√	√	√	√
EBFP2	Ai et al., Biochemistry, 2007, 46, 5904	383	448	0.60	monomer		√	√	√	√	√
SuperGlo BFP	www.qbiogene.com	387	450	n/a	monomer		√	√	√	√	√
Cyan Fluorescent Proteins
Cerulean	Rizzo et al., Nat. Biotechnol., 2004, 22, 445	433	475	0.79	monomer		√	√	√	√	√
ECFP	www.clontech.com	439	476	0.39	monomer		√	√	√	√	√
CyPet	Nguyen et al., Nat. Biotechnol., 2005, 23, 355	435	477	0.53	monomer		√	√	√	√	√
TagCFP	www.evrogen.com	458	480	0.84	dimer		√ *	√	√	√ *	√ *
mTFP1	Ai et al., Biochem. J., 2006, 400, 531	462	492	1.58	dimer		√ *	√	√	√ *	√ *
Midoriishi Cyan	www.mblintl.com	472	495	0.73	dimer		√ *	√	√	√ *	√ *
Green Fluorescent Proteins
Azami Green	www.mblintl.com	492	505	1.21	monomer	√	√	√	√	√	√
ZsGreen	www.clontech.com	493	505	1.17	tetramer	√	√	√	√	√	√
TagGFP	www.evrogen.com	482	505	1.00	monomer	√	√	√	√	√	√
EGFP	www.clontech.com	484	507	1.00	monomer	√	√	√	√	√	√
Emerald	Cubitt et al., Methods Cell. Biol., 1999, 58, 19	487	509	1.16	monomer	√	√	√	√	√	√
T-Sapphire	Zapata-Hommer et al., BMC Biotechnol., 2003, 3, 5	399	511	0.78	monomer		√	√	√	√	√
Yellow Fluorescent Proteins
TagYFP	www.evrogen.com	508	524	1.37	monomer	√	√	√	√	√	√
EYFP	www.clontech.com	514	527	1.51	monomer	√	√	√	√	√	√
Topaz	Cubitt et al., Methods Cell. Biol., 1999, 58, 19	514	527	1.69	monomer	√	√	√	√	√	√
Venus	Nagai et al., Nat. Biotechnol., 2002, 20, 87	515	528	1.56	monomer	√	√	√	√	√	√
mCitrine	Griesbeck et al., J. Biol. Chem., 2001, 276, 29188	516	529	1.74	monomer	√	√	√	√	√	√
YPet	Nguyen et al., Nat. Biotechnol., 2005, 23, 355	517	530	2.38	monomer	√	√	√	√	√	√
TurboYFP	www.evrogen.com	525	538	1.65	dimer	√	√	√	√	√	√
Orange Fluorescent Proteins
Kusabira Orange	www.mblintl.com	548	559	0.92	monomer			√	√		√
mOrange	Shaner et al., Nat. Biotechnol., 2004, 22, 1524	548	562	1.46	monomer			√	√		√
cOFP	www.stratagene.com	552	564	n/a	tetramer			√	√		√
Red Fluorescent Proteins
TurboRFP	www.evrogen.com	553	574	1.24	dimer			√	√	√	√
dTomato	Shaner et al., Nat. Biotechnol., 2004, 22, 1524	554	581	1.42	dimer			√	√	√	√
DsRed	www.clontech.com	558	583	1.76	tetramer			√	√	√	√
DsRed- Express (T1)	www.clontech.com	555	584	0.58	tetramer			√	√	√	√
TagRFP	Merzlyak et al., Nat. Methods, 2007, 4, 555 www.evrogen.com	555	584	1.46	monomer			√	√	√	√
DsRed- Monomer	www.clontech.com	556	586	0.10	monomer			√	√	√	√
mStrawberry	Shaner et al., Nat. Biotechnol., 2004, 22, 1524	574	596	0.78	monomer			√	√	√	√
mCherry	Shaner et al., Nat. Biotechnol., 2004, 22, 1524	587	610	0.47	monomer			√	√	√	√
mKeima	Kogure et al., Nat. Biotechnol., 2006, 24, 577	440	620	0.12	monomer		√ *	√ *	√ *	√ *	√ *
Far-Red Fluorescent Proteins
mRaspberry	Wang et al., PNAS, 2004, 101, 16745	598	625	0.37	monomer				√
Katushka	Shcherbo et al., Nat. Methods, 2007, 4, 741	588	635	0.67	dimer				√
mKate (TagFP635)	Shcherbo et al., Nat. Methods, 2007, 4, 741 www.evrogen.com	588	635	0.45	monomer				√
HcRed	Gurskaya et al., FEBS Lett., 2001, 507, 16	592	645	0.05	tetramer				√
mPlum	Wang et al., PNAS, 2004, 101, 16745	590	649	0.12	monomer				√
AQ143	Shkrob et al., Biochem. J., 2005, 392, 649	595	655	0.11	tetramer				√

PA-GFP	Patterson et al., Science, 2002, 297, 1873
before activation		400	515	0.08	monomer		√	√	√	√	√
after activation		504	517	0.42	monomer		√	√	√	√	√
PS-CFP2	www.evrogen.com
before activation		400	470	0.26	monomer		√	√	√	√	√
after activation		490	511	0.33	monomer		√	√	√	√	√
Dronpa	www.mblintl.com
before activation		n/a	n/a	<0.01	monomer	√	√	√	√	√	√
after activation		503	518	2.45	monomer	√	√	√	√	√	√
Kaede	www.mblintl.com
before activation		508	518	2.64	tetramer		√	√	√	√	√
after activation		572	580	0.60	tetramer			√	√	√	√
mEosFP	Weidemann et al., PNAS, 2004, 101, 15905
before activation		505	516	1.30	monomer	√	√	√	√	√	√
after activation		569	581	0.70	monomer			√	√	√	√
Dendra2	www.evrogen.com
before activation		490	507	0.45	monomer	√	√	√	√	√	√
after activation		553	573	0.39	monomer			√	√	√	√
KFP-Red	www.evrogen.com
before activation		n/a	n/a	<0.01	tetramer	n/a	n/a	n/a	n/a	n/a	n/a
after activation		580	600	0.13	tetramer			√	√	√	√
* 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

Fluorescent Proteins

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

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