Red fluorescent protein TagRFP

- Bright red (orange) fluorescence
- Fast maturation
- High pH stability
- Monomeric protein with successful performance in fusions
- Recommended for protein labeling

TagRFP is a monomeric red 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. It can also be used for cell and organelle labeling and for tracking the promoter activity; however, TurboRFP is preferable for these applications as it is brighter and more photostable than TagRFP.

Main properties

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

CHARACTERISTIC *Brightness is a product of extinction coefficient and quantum yield, divided by 1000. Molecular weight, kDa 27 Polypeptide length, aa 231 Fluorescence color red (orange) Excitation maximum, nm 555 Emission maximum, nm 583 Quantum yield 0.48 Extinction coefficient, M-1cm-1 102 000 Brightness* 49.0 Brightness, % of EGFP 148 pKa 3.8 Structure monomer Aggregation no Maturation rate at 37°C fast Photostability medium 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.# AB231-AB232) 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. It becomes clearly detectable in mammalian cells as early as within 10-12 hrs after transfection. No cell toxic effects and visible protein aggregation are observed.

	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. Cell lines expressing TagRFP fusions are commercially available.

		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 other fluorescent proteins of cyan, green, yellow, and far-red colors.

		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).

Available variants and fusions

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.

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 fluorescent labeling of Golgi apparatus.

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 fluorescent labeling of mitochondria.

TagRFP-actinin fusion: Human α-actinin is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of α-actinin in living cells.

TagRFP-tubulin fusion: Human α-tubulin is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of α-tubulin in living cells.

TagRFP-integrin fusion: Human α-V-integrin is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of α-V-integrin in living cells.

TagRFP-actin fusion: Human β-actin is linked to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of β-actin in living cells.

TagRFP-FAK fusion: Chicken focal adhesion kinase is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of focal adhesion kinase in living cells.

TagRFP-keratin fusion: Human cytokeratin-18 is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of cytokeratin-18 in living cells.

TagRFP-EB3 fusion: Human microtubule-associated EB3 protein is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of EB3 protein in living cells.

TagRFP-profilin fusion: Mouse profilin is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of profilin in living cells.

TagRFP-vinculin fusion: Human vinculin is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of vinculin in living cells.

TagRFP-zyxin fusion: Human zyxin is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of zyxin in living cells.

TagRFP-Cx26 fusion: Rat connexin 26 is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of connexin 26 in living cells.

TagRFP-Cx32 fusion: Human connexin 32 is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of connexin 32 in living cells.

TagRFP-Cx43 fusion: Rat connexin 43 is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of connexin 43 in living cells.

TagRFP-H2B fusion: Human histone H2B is fused to the TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of histone H2B in living cells.

TagRFP-laminB1 fusion: Human lamin B1 is fused to the TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of lamin B1 in living cells.