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

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).