Red Fluorescent Proteins (RFP s)/Photoactivatable
â€¢ Monomer, successful performance in fusions
â€¢ Non-fluorescent before photoactivation
â€¢ Irreversible photoactivation to a red fluorescent form by UV-violet light irradiation
â€¢ High brightness and photostability
â€¢ Recommended for monitoring of protein movement with 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].
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.
(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 2Âµm.
(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.
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 give bright fluorescent signals upon UV-activation of PA-TagRFP in 10-12 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed.
Recommended Filter Sets and Laser Lines
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.
Subach FV et al. 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
Kindling Red Fluorescent Protein (KFP-Red)
â€¢ Reversible or irreversible photoactivation
â€¢ Activated by green light that does not damage cells and tissues
â€¢ Quenching by blue light
â€¢ Recommended for tracking cells and cellular organelle movements
KFP-Red (also referred to as KFP1) is a photoactivatable GFP-like protein generated on the basis of Anemonia sulcata chromoprotein, asFP595 [Lukyanov et al., 2000; Chudakov et al., 2003a; Chudakov et al., 2003b]. KFP-Red switches from a non-fluorescent to a red fluorescent form (with excitation/emission maxima at 580 nm and 600 nm, respectively) under the exposure to intense green light irradiation. A green light laser does not damage cells and tissues. Activated KFP-Red can be easily detected because its emission spectrum is beyond the region of cell autofluorescence.
KFP-Red can be used for in vivo monitoring cell and cellular organelle movement.
Performance and Use
KFP-Red was successfully expressed and tested in various experimental models, including bacteria, Xenopus embryo, and cultured mammalian cells. Like other Anthozoa GFP-like proteins, KFP-Red is a tetramer. This restricts the wide use of KFP-Red as a fusion partner for cellular proteins.
KFP-Red reversible kindling and relaxation.
(A) Kindling and relaxation kinetics. Zero time is set at the commencement of irradiation with kindling light (532 nm laser light, 1% power). Kindling irradiation was stopped after 2 min. (B) Reversible photoactivation of KFP-Red in E. coli. The round-shaped part of the E. coli colony exp-ressing KFP-Red was irreversibly kindled with intense green light. This region fluoresces brightly, while its absorption is low. After several minutes, the kindled protein relaxed to the non-fluorescent state, while its absorption recovered.
KFP-Red irreversible kindling.
(A) Kindling kinetics. Zero time is set at the commencement of irradiation with kindling light (532 nm laser light, 20% power). Kindling irradiation was stopped after 20 min. (B) Irreversibly kindled (red line) and "unkindled" (blue line) KFP-Red fluorescence spectra and brightness ratio. The photo shows intact and irreversibly kindled KFP-Red samples after a year of incubation at room temperature.
Recommended Filter Sets and Laser Lines
KFP-Red is non-fluorescent before light activation. Upon green-light irradiation, the protein kindles to its red fluorescent form. Green light of low intensity (e.g. 1% power scanning green laser, HeNe laser line 543 nm, 1 mW, scan per 10 seconds; the number of scans is not limited) does not cause kindling and may be used as excitation light for KFP-Red visualization.
Scanning with about 5-10% power laser results in reversible kindling of KFP-Red. More intensive-light irradiation is required for irreversible KFP-Red kindling (e.g. irradiation for 20 seconds in fast mode with a 30% power green laser light induces irreversible kindling of KFP-Red in mitochondria within the irradiated field). Irradiation with weak blue laser light causes instantaneous quenching of reversibly kindled KFP-Red, whereas for the irreversibly kindled KFP-Red, quenching is not so pronounced.
TRITC filter set or similar can be used for visualization of activated KFP-Red. Omega Optical filter sets QMAX-Red and XF174 are recommended.
Kindling effect depends on temperature. Light intensity required for kindling goes down when the temperature decreases and goes up when the temperature rises. This property can be used to achieve kindling at lower light intensities by sample cooling.
Lukyanov KA et al. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem. 2000; 275 (34):25879-82. / pmid: 10852900
Chudakov DM at al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol. 2003a; 21 (2):191-4. / pmid: 12524551
Chudakov DM et al. Chromophore environment provides clue to "kindling fluorescent protein" riddle. J Biol Chem. 2003b; 278 (9):7215-9. / pmid: 12496281
PA-TagRFP-C vector: For Fusion Proteins
PA-TagRFP-N vector: For Fusion Proteins
PA-TagRFP-actin: Actin targeted PA-TagRFP
PA-TagRFP-histone H2B: Histone targeted PA-TagRFP
PA-TagRFP-tubulin: Tubulin targeted PA-TagRFP
pKindling-Red-N vector: For Fusion Proteins
pKindling-Red-B vector: Source for KFP-Red Coding Sequence
pKindling-Red-Mito: Mitochondria targeted KFP-Red
Kindling Red Properties and further application examples