TagFP635

Far-red fluorescent protein TagFP635

Newer version is available, please see mKate2 description.

 

- Bright far-red fluorescence
- Monomer with successful performance in fusions
- High photostability
- Fluorescent signal is easily distinguished from background fluorescence
- Recommended for protein labeling

TagFP635 (scientific name mKate) is a monomeric far-red fluorescent protein generated from the wild-type RFP from sea anemone Entacmaea quadricolor [Shcherbo et al., 2007]. It possesses bright fluorescence with excitation/emission maxima at 588 and 635 nm, respectively.

Main properties

TagFP635 spectra

TagFP635 normalized excitation (thin line) and emission (thick line) spectra.

Download TagFP635 spectra (xls)

CHARACTERISTIC
* Brightness is a product of extinction coefficient and quantum yield, divided by 1000.
**First value – measured at pH=8.2 [Shcherbo et al., 2007], second value – measured at pH=7.5 [Shcherbo et al., 2009].
Molecular weight, kDa27
Polypeptide length, aa237
Fluorescence colorfar-red
Excitation maximum, nm588
Emission maximum, nm635
Quantum yield0.33   /   0.28 **
Extinction coefficient, M-1cm-145 000   /   31 500 **
Brightness*14.9   /   8.8 **
Brightness, % of EGFP45   /   26 **
pKa6.0
Structuremonomer
Aggregationno
Maturation rate at 37°Cfast
Photostabilityhigh
Cell toxicitynot observed
Main advantagesmonomeric far-red fluorescent protein, high photostability

Gel-filtration of TurboGFP (dimer, green line), EGFP (monomer, blue line), and TagFP635 (monomer, red line).

Image from Shcherbo et al., 2007.

Recommended filter sets and antibodies

TagFP635 can be recognized using Anti-tRFP antibody (Cat.# AB233-AB234) available from Evrogen.

Recommended Omega Optical filter sets are QMAX-Red and XF102-2. TagFP635 can also be detected using Texas Red filter sets or similar.

Performance and use

TagFP635 can be easily expressed and detected in a wide range of organisms. Mammalian cells transiently transfected with TagFP635 expression vectors produce bright fluorescence in 12-14 hrs after transfection. No cell toxic effects and visible protein aggregation are observed.

TagFP use for cell labeling.

Transiently transfected mamalian cells expressing TagFP635.

Image of Phoenix Eco cells is from Shcherbo et al., 2007; image of a HeLa cell was kindly provided by Michael W. Davidson (Florida State University).

TagFP635 performance in fusions has been demonstrated in α-actinin, vinculin, zyxin, β-actin, α-tubulin, and other models.

TagFP635 use for protein labeling in mammalian cells.

Microscopic images of HeLa cells transiently transfected with TagFP635-tagged fusions. Arrows indicate gap junction.

Images were kindly provided by Michael W. Davidson (Florida State University).

TagFP635 can be used in multicolor labeling applications with blue, cyan, green, yellow, and red (orange) fluorescent dyes.

TagFP635 use in multicolor labeling of mammalian cells.

(A) TagCFP-tagged β-actin (cyan), TagYFP-tagged α-tubulin (green), TagFP635-H2B fusion (red), and Golgi-targeted TagRFP (orange); (B) TagCFP-tagged α-tubulin (cyan), TagFP635-clathrin fusion (red), mitochondria-targeted TagYFP (green). Images were kindly provided by Michael W. Davidson (Florida State University).


Available variants and fusions
VariantDescriptionRelated vectorCat.#Click for image
TagFP635-Cx26 fusion Rat connexin 26 is fused to the TagFP635 N-terminus. When expressed in mammalian cells, this fusion provides far-red fluorescent labeling of connexin 26 in living cells. pTagFP635-Cx26 FP382
TagFP635-Cx32 fusion Human connexin 32 is fused to the TagFP635 N-terminus. When expressed in mammalian cells, this fusion provides far-red fluorescent labeling of connexin 32 in living cells. pTagFP635-Cx32 FP383
TagFP635-Cx43 fusion Rat connexin 43 is fused to the TagFP635 N-terminus. When expressed in mammalian cells, this fusion provides far-red fluorescent labeling of connexin 43 in living cells. pTagFP635-Cx43 FP384
TagFP635-vinculin fusion Human vinculin is fused to the TagFP635 C-terminus. When expressed in mammalian cells, this fusion provides far-red fluorescent labeling of vinculin in living cells. pTagFP635-vinculin FP388


References:

  • Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, Solovieva EA, Lukyanov KA, Bogdanova EA, Zaraisky AG, Lukyanov S, Chudakov DM. Bright far-red fluorescent protein for whole-body imaging. Nat Methods. 2007; 4 (9):741-6. / pmid: 17721542
  • Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM. Far-red fluorescent tags for protein imaging in living tissues. Biochem J. 2009; 418 (3):567-74. doi: 10.1042/BJ20081949 / pmid: 19143658
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