Photoactivatable red fluorescent protein PA-TagRFP
- Monomer, successful performance in fusions
- Non-fluorescent before photoactivation
- Irreversible photoactivation to a red fluorescent form by UV-violet light irradiation
- High brightness and photostability
- Recommended for super-resolution imaging
PA-TagRFP is a photoactivatable mutant of the bright monomeric red fluorescent protein TagRFP [Subach et al., 2010]. PA-TagRFP is capable of irreversible photoconversion from non-fluorescent to red fluorescent form (with excitation/emission maxima at 562 nm and 595 nm, respectively) in response to UV-violet light irradiation.
High brightness, photostability and monomeric nature of PA-TagRFP make it an excellent protein tag for both conventional microscopy and super-resolution PALM imaging techniques [Subach et al., 2010].
Normalized excitation (thin line) and emission (thick line) spectra for activated PA-TagRFP.
Download PA-TagRFP spectra (xls)
|CHARACTERISTIC||before / after photoactivation|
|* Brightness is a product of extinction coefficient and quantum yield, divided by 1000.|
|Fluorescence color||NO / red|
|Excitation maximum, nm||- / 562|
|Emission maximum, nm||- / 595|
|Quantum yield||nd / 0.38|
|Extinction coefficient, M-1cm-1||nd / 66 000|
|Brightness*||0 / 25.1|
|pKa||nd / 5.3|
|Activating light||UV-violet (e.g. 390-420 nm)|
|Calculated contrast, fold||~540|
|Cell toxicity||not observed|
|Maturation rate at 37°C||fast|
|Molecular weight, kDa||27|
|Polypeptide length, aa||237|
Recommended antibodies, filter sets and laser lines
PA-TagRFP can be recognized using Anti-tRFP antibody (Cat.# AB233-AB234) available from Evrogen.
PA-TagRFP is non-fluorescent before light activation. Upon UV-violet irradiation the protein irreversibly converts to its red fluorescent form.
PA-TagRFP can be activated during both widefield imaging (e.g. the Arc-lamp irradiation, 100xoil objective, 390-420 nm, 10-50 mW/cm2) and confocal laser scanning imaging (e.g. 405 nm laser line, estimated < 2.5 W/cm2 at the sample). Maximal efficiency of photoactivation for PA-TagRFP is observed at 390-420 nm. The photoactivation efficiency drops dramatically with the wavelength increasing above 420 nm.
The source of irradiation, irradiation time and intensity of activating UV-violet light must be individually adjusted for particular instrumentation and intended application.
TRITC filter set or similar can be used for visualization of activated PA-TagRFP. Omega Optical filter sets QMAX-Red and XF174 are recommended.
Performance and use
PA-TagRFP can be easily expressed and detected in a wide range of organisms. Mammalian cells transiently transfected with PA-TagRFP expression vectors produce bright fluorescence upon UV-activation of PA-TagRFP in 10-12 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed.
PA-TagRFP use for cell labeling.
Live HeLa cells transiently transfected with the PA-TagRFP-C expression vector were imaged during the photoactivation.
PA-TagRFP performance in protein fusions has been demonstrated in β-actin, α-tubulin, histone H2B and other models.
PA-TagRFP use in PALM imaging techniques
High brightness, photostability and absence of initial fluorescence signal from PA-TagRFP make it a protein tag of choice for super resolution two-color PALM/single-particle tracking PALM imaging techniques. The excellent performance of PA-TagRFP in two-color single-particle tracking PALM experiments was demonstrated for several PA-TagRFP-tagged and PAGFP-tagged fusions in live COS-7 cells [Subach et al., 2010].
An example for the tracking of PA-TagRFP-tagged epidermal growth factor receptor (EGFR-PATagRFP) and PAGFP-tagged vesicular stomatitus virus G protein tsO45 (VSVG-PAGFP) in live COS-7 cells by two-color single-particle tracking PALM is shown below.
(G) A zoomed view of the region indicated by the square in (F).
Available variants and fusions
PA-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.
Human β-actin is fused to the PA-TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of β-actin in living cells.
Human α-tubulin is fused to the PA-TagRFP C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of α-tubulin in living cells.
Human histone H2B is fused to the PA-TagRFP N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of histone H2B in living cells.
Haas J, Park EC, Seed B.
Codon usage limitation in the expression of HIV-1 envelope glycoprotein.
Curr Biol. 1996; 6 (3):315-24. / pmid: 8805248
Subach FV, Patterson GH, Renz M, Lippincott-Schwartz J, Verkhusha VV.
Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
J Am Chem Soc. 2010; 132 (18):6481-91. doi: 10.1021/ja100906g / pmid: 20394363