Fluorescent proteins are an important part of any molecular biologist’s toolkit. Recombinant DNA techniques allow researchers to incorporate a fluorescent protein into an engineered plasmid and track its expression over time with flow cytometry or, more commonly, fluorescent microscopy methods. Here we explore the history, advantages, common uses and wide selection of fluorescent proteins.

 

History

The bioluminescent proteins green fluorescent protein (GFP) and aequorin were first isolated from the jellyfish species Aequorea Victoria in 1961 (PMID: 13911999). However, this discovery remained fairly unknown until the gene coding for GFP was sequenced and cloned in 1992 (PMID: 1347277). Subsequent papers demonstrated the many potential research applications for GFP (PMID: 8682203), leading to the widespread use of recombinant fluorescent probes to observe a wide variety of cellular processes in living systems.

 

Advantages

Fluorescent proteins offer several advantages over alternative fluorescent molecules.

  • Fluorescent proteins do not require fixation, enzymes, or substrates to generate fluorescence.
  • While FITC is highly phototoxic, illuminating GFP is generally not harmful to living cells. This allows researchers to observe cells over time, without affecting their long-term viability.
  • When introduced properly, fluorescent-tagged proteins can be continually expressed and even passed on to subsequent generations of cells, allowing for longer-term studies of protein expression in cells or tissue. This heritability has even led to the creation of countless stable GFP-expressing cell lines and transgenic animal models.
  • Fluorescent protein expression can be specifically controlled via inducible promoters. This allows researchers to activate GFP expression at the exact time required for their experiment.

 

Applications

Fluorescent proteins are most often used in reporter assays to identify if/when a specific gene is being expressed by a cell, tissue or organism. A few common examples include:

  • As transformational reporters. Inserting a fluorescent protein gene into the same DNA construct being introduced to cells allows researchers to visually confirm that their transformation was successful. FACS analysis can then be used to enrich populations for positively transformed cells.
  • As direct transcriptional and translational reporters
  • As quantitative reporters of gene expression (PMID: 15640280)

They are also useful for mapping gene expression during cellular interactions or tissue development (PMID: 14671301)

 

Colors

Random and directed mutagenesis efforts on the wild type GFP have generated new genetic variants with enhanced brightness (EGFP) (PMID: 8707053) and folding characteristics (sfGFP) (PMID: 16369541), as well as many spectral variants ranging from yellow (YFP) (PMID: 11753368) to blue (EBFP) and cyan (ECFP).

Additional naturally occurring fluorescent proteins have been identified in other marine species from the phylum Cnidaria, including reef coral Anthozoa species (PMID: 10504696) and sea anemone Discosoma species (PMID: 23000031). These include red (dsRed) and orange variants which have subsequently undergone mutagenesis efforts to improve folding and spectral characteristics.

To date, a broad range of fluorescent proteins have been discovered or developed to span the visible light spectrum (PMID: 19771335). This table details the a few of the most common fluorescent protein options available on FluoroFinder’s experiment design tool.

 

NameExcitation MaxEmission MaxMolecular WeightOligomerizationOrganismView Products
eBFP380nm440nm26.9 kDaWeak dimerAequorea victoriaView Products
BFP381nm445nm26.8 kDaDimerAequorea victoriaView Products
TagBFP402nm457nm26.3 kDaMonomerEntacmaea quadricolorView Products
mTurquoise434nm474nm26.9 kDaMonomerAequorea victoriaView Products
Cerulean433nm475nm26.8 kDaWeak dimerAequorea victoriaView Products
eCFP434nm477nm26.9 kDaMonomerAequorea victoriaView Products
CFP456nm480nm26.9 kDaDimerAequorea victoriaView Products
AmCyan-Protein453nm486nm25.3 kDaTetramerAnemonia majanoView Products
mTFP1462nm492nm26.9 kDaMonomerClavularia sp.
MiCy1472nm495nm26.2 kDaDimerAcropora sp.View Products
ZsGreen496nm506nm26.1 kDaTetramerZoanthus sp.View Products
eGFP488nm507nm26.9 kDaWeak dimerAequorea victoriaView Products
GFP395nm509nm26.9 kDaWeak dimerAequorea victoriaView Products
Emerald GFP487nm509nm26.9 kDaWeak dimerAequorea victoriaView Products
T-Sapphire399nm511nm26.9 kDaWeak dimerAequorea victoriaView Products
mAmetrine406nm526nm26.8 kDaMonomerAequorea victoriaView Products
Topaz514nm527nm26.9 kDaWeak dimerAequorea victoriaView Products
YFP514nm527nm26.9 kDaWeak dimerAequorea victoriaView Products
eYFP513nm527nm27.0 kDaWeak dimerAequorea victoriaView Products
Venus515nm528nm26.8 kDaWeak dimerAequorea victoriaView Products
mCitrine516nm529nm27.1 kDaMonomerAequorea victoriaView Products
mBanana540nm553nm26.6 kDaMonomerDiscosoma sp.View Products
mKO540nm553nm26.6 kDaMonomerDiscosoma sp.View Products
mHoneyDew487nm562nm25.4 kDaMonomerDiscosoma sp.View Products
mOrange548nm562nm26.7 kDaMonomerDiscosoma sp.View Products
mKusabira-Orange2551nm565nm24.5 kDaMonomerVerrillofungia concinnaView Products
OFP548nm573nm25.1 kDaTetramerCerianthariaView Products
tdTomato554nm581nm54.2 kDaTandem dimerDiscosoma sp.View Products
dTomato554nm581nm27.0 kDaDimerDiscosoma sp.View Products
RFP558nm583nm25.9 kDaTetramerDiscosoma sp.View Products
dsRed558nm583nm25.9 kDaTetramerDiscosoma sp.View Products
TagRFP657555nm584nm26.1 kDaWeak dimerEntacmaea quadricolorView Products
mTangerine568nm585nm25.4 kDaMonomerDiscosoma sp.View Products
DsRed Express554nm586nm25.7 kDaTetramerDiscosoma sp.View Products
DsRed2561nm587nm25.8 kDaTetramerDiscosoma sp.View Products
mApple568nm592nm27.0 kDaMonomerDiscosoma sp.View Products
mStrawberry574nm596nm26.6 kDaMonomerDiscosoma sp.View Products
LSS-mKate2460nm605nm26.2 kDaMonomerEntacmaea quadricolorView Products
mRuby558nm605nm25.2 kDaMonomerEntacmaea quadricolorView Products
mRFP584nm607nm25.4 kDaMonomerDiscosoma sp.View Products
mCherry587nm610nm26.7 kDaMonomerDiscosoma sp.View Products
mKeima440nm620nm25.1 kDaMonomerMontipora sp.View Products
LSS-mKate1463nm624nm26.2 kDaMonomerEntacmaea quadricolorView Products
mRaspberry598nm625nm25.5 kDaMonomerDiscosoma sp.View Products
Katushka2S588nm633nm26.4 kDaDimerEntacmaea quadricolorView Products
mKate2588nm633nm26.1 kDaMonomerEntacmaea quadricolorView Products
Hc Red592nm645nm25.6 kDaTetramerHeteractis crispaView Products
E2-Crimson611nm646nm25.7 kDaTetramerDiscosoma sp.View Products
mPlum590nm649nm25.6 kDaMonomerDiscosoma sp.View Products
TagRFP657611nm657nm26.3 kDaMonomerEntacmaea quadricolorView Products
NirFP605nm670nm26.0 kDaDimerEntacmaea quadricolorView Products
iRFP 670643nm670nm34.5 kDaDimerRhodopseudomonas palustrisView Products
iRFP 682663nm682nm34.5 kDaDimerRhodopseudomonas palustrisView Products
iRFP 702673nm702nm34.5 kDaDimerRhodopseudomonas palustrisView Products
IFP 1.4696nm708nm34.8 kDaMonomerDeinococcus radioduransView Products
iRFP 713690nm713nm34.6 kDaDimerRhodopseudomonas palustrisView Products
iRFP 720702nm720nm34.6 kDaDimerRhodopseudomonas palustrisView Products

 

Fluorescent proteins are a powerful tool for researchers monitoring gene expression or performing any molecular cloning experiments. These proteins offer many unique advantages over alternative fluorescent or colorimetric reporter assays, and the discovery and development of new fluorescent proteins have further expanded this important toolkit.