Far red proteins avoid the natural green autofluorescence found in plants and animals, and are preferred for in vivo imaging.
E2-Crimson
E2-Crimson was derived from DsRed-Express2, and retains DsRed-Express2's fast maturation, high photostability, high solubility, and low cytotoxicity (1). It is well-suited for in vivo applications involving sensitive cells such as primary cells and stem cells:
Extremely bright
Very soluble
Fastest maturing red or far red—half time of 26 minutes at 37°C
Efficient excitation with standard far red lasers
Suitable for multicolor experiments
Figure 1. E2-Crimson is useful for confocal and STED (stimulated emission depletion) microscopy. The mammalian ER was imaged by conventional confocal microscopy (left) or by STED microscopy (right) with 635 nm excitation and a STED wavelength of 760 nm. The scale bar is 1 µm.
mPlum & mRaspberry
Clontech is distributing vectors encoding several of the Fruit Fluorescent Proteins, including mPlum and mRaspberry, which were developed in the laboratory of Dr. Roger Tsien (2–4). They are mutants derived from mRFP1, a monomeric mutant of DsRed, by directed mutagenesis (5). They have demonstrated stable expression, perform successfully in numerous fusion applications, and are already well characterized and recognized in the literature.
Effective Fusion Constructs
mPlum and mRaspberry are well-tolerated as fusion proteins in a wide variety of applications. These fusions have also been used for quantitative imaging techniques including fluorescence resonance energy transfer (FRET).
HcRed1
HcRed is a far-red fluorescent protein derived from a nonfluorescent chromoprotein found in the Anthozoa-class sea anemone Heteractis crispa. It was generated using random and site-directed mutagenesis (6). The HcRed1 coding sequence is human codon-optimized for enhanced expression in mammalian cells. HcRed1 has a low tendency to form aggregates in living cells, and can be detected just 16 hours after transfection by flow cytometry, a time frame which is comparable with EGFP.
Living Colors Far Red Fluorescent Proteins
Protein
Color
Excitation Maximum (nm)
Emission Maximum (nm)
Quantum Yield
Extinction Coefficient (M-1cm-1)
Brightness
mPlum
far red
590
649
0.10
41,000
4,100
E2-Crimson
far red
611
646
0.23
126,000
28,900
mRaspberry
far red
598
625
0.15
86,000
12,900
HcRed1
far red
588
618
0.03
20,000
600
Quantum Yield measurements were performed using the equation QYFP = (Qfluo * FFP * ODfluo) / (Ffluo * ODFP)
where F is the fluorescence and OD is the optical density of either the fluorescent protein (FP) or the reference fluorophore (fluo)
Molar Extinction Coefficient (Ε) was determined as follows. These molar extinction coefficient numbers are at their respective absorption maxima.
A = Ε * C * l (Beer & Lambert law) where A=absorbance, C=conc, l=path length (1cm)
or Ε = A/C (C determined by Bradford method) Brightness = Quantum Yield multiplied by Extinction Coefficient
References
Strack, R. L. et al. (2009) Biochemistry 48(35):8279–8281.
Shaner, N. C. et al. (2004) Nature Biotechnol.22(12):1567–1572.
Wang, L. et al. (2004) Proc. Nat. Acad. Sci.101(48):16745–16749.
Shu, X. et al. (2006) Biochemistry 45(32):9639–9647.
Campbell, R. E. et al. (2002) Proc. Nat. Acad. Sci.99(12):7877–7882.
Gurskaya, N. G., et al. (2001) FEBS Letters507(1):16–20.
