* Brightness is a product of extinction coefficient and quantum yield, divided by 1000.


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 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).

References:

1. 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
2. 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. / pmid: 20394363

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