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.

Recommended for Flow Cytometry Fluorescent Proteins

Protein Names	Reference or Source	Spectral Properties			Oligomeric State	AECOM core facility flow cytometer
		Peak Excitation nm	Peak Emission nm	Brightness (relative to eGFP)		FACScan	LRSII	MoFlo	MoFlo XDP	FACSAria	Forcheimer FACSAria
Blue Fluorescent Proteins
Sirius	Tomosugi et at., Nat. Methods, 2009, 6, 351-353	355	424	0.11	monomer		✓	✓	✓	✓	✓
EBFP2	Ai et al., Biochemistry, 2007, 46, 5904	383	445	0.60	monomer		✓	✓	✓	✓	✓
Azurite	Mena et al., Nat. Biotechnol., 2006, 24, 1569	383	448	0.43	monomer		✓	✓	✓	✓	✓
TagBFP	Subach et al., Chem Biol, 2008, 59, 116-1124 www.evrogen.com	400	456	0.99	monomer		✓	✓	✓	✓	✓
Cyan Fluorescent Proteins
mTarquoise	Goedhart et al., Nat. Methods, 2010, 7, 137-139	434	474	0.75	monomer		✓	✓	✓	✓	✓
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		✓	✓	✓	✓	✓
mTFP1	Ai et al., Biochem. J., 2006, 400, 531	462	492	1.58	dimer		✓ *	✓	✓	✓ *	✓ *
Green Fluorescent Proteins
TagGFP2	www.evrogen.com	482	505	1	monomer	✓	✓	✓	✓	✓	✓
EGFP	www.clontech.com	484	507	1	monomer	✓	✓	✓	✓	✓	✓
Emerald	Cubitt et al., Methods Cell. Biol., 1999, 58, 19	487	509	1.16	monomer	✓	✓	✓	✓	✓	✓
Superfolder GFP	Pedelacq et al., Nat. Biotechnol, 2006, 24, 79-88	485	510	1.6	monomer	✓	✓	✓	✓	✓	✓
Yellow Fluorescent Proteins
EYFP	www.clontech.com	514	527	1.51	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
mKO	Karasawa, S., et al., Biochem J, 2004, 381, 307-312 www.mblintl.com	548	559	0.92	monomer			✓	✓		✓
E2-Orange	Strack et al., BMC Biotechnol, 2009, 9, 32	540	561	0.61	tetramer			✓	✓		✓
mOrange	Shaner et al., Nat. Biotechnol., 2004, 22, 1524	548	562	1.46	monomer			✓	✓		✓
mKOk	Tsutsui H, et al., Nat. Methods 2008, 5, 683-685	551	563	1.9	monomer			✓	✓		✓
Red Fluorescent Proteins
dTomato	Shaner et al., Nat. Biotechnol., 2004, 22, 1524	554	581	1.42	dimer			✓	✓	✓	✓
TagRFP	Merzlyak et al., Nat. Methods, 2007, 4, 555 www.evrogen.com	555	584	1.46	monomer			✓	✓	✓	✓
DsRed- Express2	Strack et al., Nat Methods. 2008, 5, 955-957 www.clontech.com	554	591	0.45	tetramer			✓	✓	✓	✓
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			✓	✓	✓	✓
Far-Red Fluorescent Proteins
Katushka2	Shcherbo et al., Biochem J, 2009, 418, 567-574 www.evrogen.com	588	635	0.73	dimer				✓
mKate2	Shcherbo et al., Biochem J, 2009, 418, 567-574 www.evrogen.com	588	633	0.74	monomer				✓
E2- Crimson	Strack et al., Biochem, 2009, 48, 8279-8281 www.clontech.com	611	646	0.86	tetramer				✓	✓	✓
eqFP650	Shcherbo et al., Nat. Methods, 2010	592	650	0.46	dimer				✓
mNeptune	Lin et al., Chem Biol, 2009, 16, 1169-79	600	650	0.40	monomer				✓
Near-infrared Fluorescent Proteins
eqFP670	Shcherbo et al., Nat. Methods, 2010	605	670	0.12	dimer				✓
TagRFP657	Morozova et al., Biophys J, 2010, 99, L13-L15	611	657	0.10	monomer				✓	✓	✓
iRFP	Filonov et al., Nat Biotechnology, 2011, 29, 757–76	690	713	0.18	dimer			✓	✓
Large Stokes Shift Green and Red Flurescent Proteins
T-Sapphire	Zapata-Hommer et al., BMC Biotechnol., 2003, 3, 5	399	511	0.78	monomer		✓	✓	✓	✓	✓
mAmertrine	Ai et al., Nat Methods, 2008, 5, 401-403	406	526	0.78	monomer		✓	✓	✓	✓	✓
LSSmKate2	Piatkevich et al., PNAS, 2010, 107, 5369-5374	460	605	0.13	monomer		✓ *	✓ *	✓	✓	✓
mKeima	Kogure et al., Nat. Biotechnol., 2006, 24, 577 www.mblintl.com	440	620	0.10	dimer		✓ *	✓ *	✓	✓	✓
Flurescent Timers that change					color from Blue to Red with time
Slow-FT	Subach et al., Nat Chem Biol, 2009, 5, 118-126
blue form		402	465	0.35	monomer		✓	✓	✓	✓	✓
red form		583	604	0.13				✓	✓	✓	✓
Medium-FT	Subach et al., Nat Chem Biol, 2009, 5, 118-126
blue form		401	464	0.55	monomer		✓	✓	✓	✓	✓
red form		579	600	0.17				✓	✓	✓	✓
Fast-FT	Subach et al., Nat Chem Biol, 2009, 5, 118-126
blue form		403	466	0.44	monomer		✓	✓	✓	✓	✓
red form		583	606	0.20				✓	✓	✓	✓
mk-Go	Tsuboi et al., Mol Biol Cell, 2010, 21, 87-94
green form		500	509	n/a	monomer	✓	✓	✓	✓	✓	✓
orange form		548	561	n/a				✓	✓		✓
PA-GFP	Patterson et al., Science, 2002, 297, 1873
before activation		400	515	0.08	monomer		✓	✓	✓	✓	✓
after activation		504	517	0.42			✓	✓	✓	✓	✓
PS-CFP2	www.evrogen.com
before activation		400	470	0.26	monomer		✓	✓	✓	✓	✓
after activation		490	511	0.33			✓	✓	✓	✓	✓
Dronpa	www.mblintl.com
before activation		n/a	n/a	<0.01	monomer	n/a	n/a	n/a	n/a	n/a	n/a
after activation		503	518	2.45			✓	✓	✓	✓	✓
tdEosFP	Nienhaus et al., PNAS, 2005, 102, 9156
before activation		506	516	1.65	pseudomonomer	✓	✓	✓	✓	✓	✓
after activation		569	581	0.59				✓	✓	✓	✓
mEos2	McKinney et al., Nat Methods, 2009, 6, 131
before activation		506	519	1.4	monomer	✓	✓	✓	✓	✓	✓
after activation		573	584	0.90				✓	✓	✓	✓
Dendra2	www.evrogen.com
before activation		490	507	0.45	monomer	✓	✓	✓	✓	✓	✓
after activation		553	573	0.39				✓	✓	✓	✓
PAmCherry	Subach et al., Nat Methods, 2009, 6, 153-159
before activation		No	No	No	monomer
after activation		564	594	0.25				✓	✓	✓	✓
PATagRFP	Subach et al., J Am Chem Soc, 2010, 132, 6481-6491
before activation		No	No	No	monomer
after activation		562	595	0.75				✓	✓	✓	✓
rsTagRFP	Subach et al., Chem Biol, 2010, 17, 745-755
OFF		567	585	0.005	monomer			✓	✓	✓	✓
ON		567	585	0.12				✓	✓	✓	✓
* Excitation is suboptimal using the cytometer's existing laser lines.

General Reviews on Modern Fluorescent Proteins:
1.	Shcherbakova D.M. and Verkhusha V.V. Chromophore chemistry of fluorescent proteins controlled by light. Curr. Opin. Chem. Biol. 2014, 20: 60-68.
2.	Shcherbakova D.M. et al. Red fluorescent proteins: advanced imaging applications and future design. Angew. Chem. Int. Ed. 2012, 51: 10724-10738.
3.	Miyawaki A. et al. Red fluorescent proteins: chromophore formation and cellular applications. Curr. Opin. Struct. Biol. 2012, 22: 679-688.
4.	Chudakov D.M. et al. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 2010, 90, 1103-1163.
5.	Day R.N. and Davidson M.W. The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev. 2009, 38, 2887-921.
Use of Fluorescent Proteins in Flow Cytometry:
1.	Telford W.G. et al. Flow cytometry of fluorescent proteins. Methods 2012, 56: 318-330.
2.	Piatkevich K.D. and Verkhusha V.V. Guide to red fluorescent proteins and biosensors for flow cytometry. Methods Cell. Biol. 2011, 104: 431-461.
Internet resources and commercially available fluorescent proteins:
1.	Florida State University
2.	Clontech Laboratories
3.	Evrogen
4.	Invitrogen Molecular Probes
5.	MBL International
6.	Non-profit Addgene
With further questions on fluorescent proteins, contact Dr. Vlad Verkhusha at the Albert Einstein College of Medicine's Department of Anatomy and Structural Biology.