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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.
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. |
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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. |
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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 |
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With further questions on fluorescent proteins, contact Dr. Vlad Verkhusha at the Albert Einstein College of Medicine's Department of Anatomy and Structural Biology. |
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