Fluorescent Protein Resource Center

Preferred Fluorescent Proteins

The discovery that green fluorescent protein (GFP) variants and coral fluorescent proteins can be functionally expressed in heterogeneous systems has revolutionized cell biology. Unmodified fluorescent proteins (FPs) can be visualized by fluorescence microscopy and can serve as probes of environments within living cells. Addition of targeting and retention sequences to FPs can be exploited to highlight specific cellular organelles and to follow their dynamics. The ability of FPs to fold, even when fused to cellular proteins, has made it possible to directly study the biology of proteins in vivo. Proteins of interest can be monitored in cells or even whole animals without having to purify, label, and deliver the proteins into cells. It is now possible to label and observe proteins in previously inaccessible environments such as organelle lumena. Fusion of FPs to proteins of interest can reveal a wealth of information including a protein’s steady state distribution, dynamics, history, and association with other proteins.

To design a fluorescent fusion protein (FFP), the investigator must consider the intended use of the FFP intended use, which fluorescent tag to add, whether the FP has issues related to the protein of interest’s environment (i.e. pH sensitivity or enhanced aggregation), and where to insert the FP. The actual construction of FFPs can be readily accomplished with standard molecular biology techniques. Useful stragegies are described in this PDF [Design and Use of Fluorescent Fusion Proteins in Cell Biology].

The list of available FPs is constantly expanding [The Fluorescent Protein Color Palette] and can be daunting. Not only are there proteins with colors that range across the entire visible spectrum, but there are FPs that change color upon laser activation- photoactivatable proteins. For more on photoactivatable proteins, see this [Photoactivatable Fluorescent Proteins]. In effect, an investigator can perform an optical pulse-chase with the photoactivatable proteins. A select population of proteins in a cell can be photoactivated on a confocal laser scanning microscope and then that population of protein can be followed over time for changes in localization, degradation, etc.

Which fluorescent protein to use?

We are continuously updating the list of fluorescent proteins as new proteins are published and as we test them. No fluorescent protein is perfect. The spectrum may be too wide and overlap with another fluorophore you are using. The protein may photobleach too quickly or may be too dim. Tables of the the better known and commonly used fluorescent proteins and photoactivatable proteins. Too simplify your choices, we have assembled the table below, which lists our current preferred fluorescent proteins. Sequence files and PDF files of protein properties will be coming soon to this site. Please contact Vlad if you have any questions about which fluorescent protein is right for your application or if there is a new fluorescent protein not listed in the tables.

Useful resources: [Design and Use of Fluorescent Fusion Proteins in Cell Biology] [The Fluorescent Protein Color Palette] [Photoactivatable Fluorescent Proteins]

Protein Name	Excitation, nm	Emission, nm	Brightness relative to EGFP	Oligomeric State	Reference or Source	FP Characteristics (pdf)
Preferred Fluorescent proteins
Preferred Blue
Cerulean	433	475	0.79	monomer*	Rizzo et al., Nat. Biotechnol., 2004, 22, 445	mCerulean
Preferred green
mEGFP	484	507	1.0	monomer	Clontech, Snapp et al. J. Cell Biol. 2003, 163:25
Preferred yellow
mVenus	515	528	1.56	monomer	Nagai et al., Nat. Biotechnol., 2002, 20, 87
Preferred red
mCherry	587	610	0.47	monomer*	Shaner et al., Nat. Biotechnol., 2004, 22, 1524
Preferred photoactivatable
Blue to green
PA-GFP	505	516	1.30	monomer	Patterson et al., Science, 2002, 297, 1873
Green to red
Dendra2				monomer	www.evrogen.com
before activation	490	507	0.45
after activation	553	573	0.39
Cell Marker Proteins Not for protein fusions
tdTomato	554	581	1.42	dimer	Shaner et al., Nat. Biotechnol., 2004, 22, 1524
Photoactivatable
PS-CFP2				monomer*	www.evrogen.com
before activation	400	470	0.26
after activation	490	511	0.33
Kaede				tetramer	www.mblintl.com
before activation	508	518	2.64
after activation	572	580	0.60

* While these proteins are classified as monomers, they have a tendency to aggregate and are not recommended for making fusions with proteins known to oligomerize.
** Most fluorescent proteins are covered by patents and you must either purchase the plasmid or sign an MTA to legally use the fluorescent protein. For this reason, we are not a fluorescent protein clearing house. Proteins with a ** are available for distribution. The other proteins are also freely available- nonmonomerized CFP, GFP, YFP, monomerized CFP, YFP, and psCFP-1.