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].

Main properties

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 Structure monomer Cell toxicity not observed Aggregation no 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) 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 for protein labeling in mammalian cells. Microscopic images of HeLa cells transiently transfected with PA-TagRFP-tagged fusions after the photoactivation: (A) β-actin; (B) α-tubulin; (C) histone H2B.

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.

(A,B) The separate and (C) merged distribution of VSVG-PAGFP (green) and EGFR-PATagRFP (red) in PALM images. Arrows indicate areas of apparent colocalization between the VSVG and EGFR molecules. Scale bars are 2µm.

(D,E) Tracks of VSVG-PAGFP and EGFR-PATagRFP molecules lasting longer than 0.7 sec are plotted. Approximately 1635 VSVG molecules were tracked along with 627 EGFR molecules.(F) VSVG-PAGFP (green) and EGFR-PATagRFP (red) tracks are merged.

	(G) A zoomed view of the region indicated by the square in (F).

Available variants and fusions

Variant	Description	Related vector	Cat.#
Humanized PA-TagRFP	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.	pPA-TagRFP-C	FP811
		pPA-TagRFP-N	FP812
PA-TagRFP-tubulin fusion	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.	pPA-TagRFP-tubulin	FP814
PA-TagRFP-H2B fusion	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.	pPA-TagRFP-H2B	FP815