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)
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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.