Red fluorescent protein TagRFP

- Bright red (orange) fluorescence
- Monomeric protein with successful performance in fusions
- Fast maturation, high pH-stability
- Proven suitability to generate stably transfected cell lines
- Recommended for protein labeling, acidic organelle labeling, FRET applications

TagRFP is a monomeric red (orange) fluorescent protein generated from the wild-type RFP from sea anemone Entacmaea quadricolor [Merzlyak et al., 2007]. It possesses bright fluorescence with excitation/emission maxima at 555 and 584 nm, respectively. TagRFP is about three times brighter than mCherry protein [Shaner et al., 2004], which makes it the brightest monomeric red fluorescent protein available so far. TagRFP is mainly intended for protein labeling and FRET applications. It can also be used for cell and organelle labeling and for tracking the promoter activity.

Main properties

TagRFP normalized excitation (thin line) and emission (thick line) spectra. Spectra viewer tool Download TagRFP spectra (xls)

CHARACTERISTIC * Brightness is a product of extinction coefficient and quantum yield, divided by 1000. ** Time to bleach 50% of fluorescent signal brightness. Molecular weight, kDa 27 Polypeptide length, aa 237 Fluorescence color red (orange) Excitation maximum, nm 555 Emission maximum, nm 584 Quantum yield 0.48 Extinction coefficient, M-1cm-1 100 000 Brightness* 48.0 Brightness, % of EGFP 148 pKa 3.8 Structure monomer Aggregation no Maturation half-time, min 100 Photostability, widefield** 48 Photostability, confocal** 125 Cell toxicity not observed Main advantages bright red monomeric fluorescent protein Possible limitations medium photostability

Recommended filter sets and antibodies

TagRFP can be recognized using Anti-tRFP antibody (Cat.# AB233) available from Evrogen.

Recommended Omega Optical filter sets are QMAX-Yellow, XF108-2, XF101-2, and XF111-2. TagRFP can also be detected using TRITC filter set or similar.

Performance and use

TagRFP can be easily expressed and detected in a wide range of organisms. Mammalian cells transiently transfected with TagRFP expression vectors produce bright fluorescence in 10-12 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed. High pH-stability with pKa=3.8 makes it possible to use TagRFP for imaging in acidic organelles, such as late and recycling endosomes and lysosomes.

	TagRFP use for cell and organelle labeling. Microscopic images of HeLa cells transiently transfected with TagRFP and TagRFP-targeted to cellular organelles. Images were kindly provided by Michael W. Davidson (Florida State University).

TagRFP performance in protein fusions has been demonstrated in fibrillarin, vinculin, zyxin, β-actin, α-tubulin, and other models.

TagRFP use for protein labeling in mammalian cells. Microscopic images of HeLa cells transiently transfected with TagRFP-tagged fusions. Images were kindly provided by Michael W. Davidson (Florida State University).

TagRFP suitability to generate stably transfected cells has been proven by Marinpharm company.

		Stably transfected cell lines expressing TagRFP fusions. U-205 mammalian cell lines expressing (A) TagRFP fusion with α-tubulin and (B) TagRFP fusion with β-actin. Images were kindly provided by Dr. Christian Petzelt (Marinpharm).

TagRFP can be used in multicolor labeling applications with blue, cyan, green, yellow, and far-red fluorescent dyes.

		TagRFP use in multicolor labeling of mammalian cells. (A) TagCFP-tagged β-actin (cyan), TagYFP-tagged α-tubulin (yellow), TagFP635-H2B fusion (red), and Golgi-targeted TagRFP (violet); (B) mitochondria-targeted TagCFP (cyan), Dendra2-tagged vimentin (green), and TagRFP-tagged β-actin (red). Image (A) was kindly provided by Michael W. Davidson (Florida State University).

TagRFP is ideally suitable for use as an acceptor for FRET with Evrogen green fluorescent protein TagGFP2. The calculated Forster distance (R₀ = 5.7 nm) for the TagGFP2-TagRFP pair is one of the largest among the values reported. At the same time, since TagGFP2 and TagRFP emission peaks are spaced by as much as 78 nm, the emission signal for these two proteins can be easily separated in any imaging system. High pH-stability of the both proteins allows using this pair for imaging in acidic organelles.

The excellent performance of TagRFP in FRET application was demonstrated both in vitro and in vivo on the example of FRET-based apoptosis reporter Casper3-GR [Shcherbo et al., 2009].

Available variants and fusions

Variant	Description	Related vector	Cat.#	Click for image
Humanized TagRFP	TagRFP codon usage is optimized for high expression in mammalian cells [Haas et al., 1996], but it can be successfully expressed in many other heterological systems.	pTagRFP-C	FP141
		pTagRFP-N	FP142
TagRFP-AS variant	TagRFP-AS codon usage is optimized for high expression in Arabidopsis and Saccharomyces. This variant is available in Gateway® entry clones.	Gateway® TagRFP-AS-C entry clone	FP148
		Gateway® TagRFP-AS-N entry clone	FP149
TagRFP-actin fusion	Human β-actin is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red (orange) fluorescent labeling of β-actin in living cells.	pTagRFP-actin	FP144
TagRFP-tubulin fusion	Human α-tubulin is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red (orange) fluorescent labeling of α-tubulin in living cells.	pTagRFP-tubulin	FP145
TagRFP-mito fusion	A mitochondrial targeting sequence (MTS) is fused to the TagRFP N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. When expressed in mammalian cells, this variant provides red (orange) fluorescent labeling of mitochondria.	pTagRFP-mito	FP147
TagRFP-actinin fusion	Human α-actinin is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red (orange) fluorescent labeling of α-actinin in living cells.	pTagRFP-actinin	FP360
TagRFP-Cx43 fusion	Rat connexin 43 is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red (orange) fluorescent labeling of connexin 43 in living cells.	pTagRFP-Cx43	FP364
TagRFP-Golgi fusion	Golgi targeting sequence (fragment of human β-1,4-galactosyltransferase) is fused to the TagRFP N-terminus. When expressed in mammalian cells, this variant provides red (orange) fluorescent labeling of Golgi apparatus.	pTagRFP-Golgi	FP367
TagRFP-H2B fusion	Human histone H2B is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red (orange) fluorescent labeling of histone H2B in living cells.	pTagRFP-H2B	FP368