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EosFP was isolated from the stony coral Lobophyllia hemprichii. Initially, the protein matures in a green fluorescent state with an emission maximum at 516 nm. Upon irradiation with violet-blue light the chromophore undergoes an irreversible photoconversion to a red state emitting at 581 nm. The wavelengths required for photoconversion and detection of the green and red fluorescent states can be easily separated, making EosFP an excellent choice for regional optical marking.

Applications:

Applications of EosFP are based on the principle that the marker is regionally photoconverted from green to red. Subsequently, the red fluorescent fraction can be tracked independently. Major applications of EosFP are:

1. Tracking of cells (cell fate mapping, tracking of metastases)

EosFP is used to label cells. The protein can be introduced into the cell by transfection of expression vectors or by microinjection of in vitro transcribed mRNA or purified recombinant protein. A desired cell or cell group is labeled and can be tracked by photoconversion.

2. Tracking of subcellular compartments

EosFP is fused to a subcellular targeting signal. The organelle or parts thereof are labeled by photoconversion. The movement of the organelles can be monitored subsequently.

3. Tracking of proteins

EosFP is fused to a protein of interest. After photoconversion, the movement of the marked fraction of the protein can be followed by red fluorescence.

4. Fluorescence nanoscopy

EosFP is fused to a protein of interest. The subcellular localization can be determined with a resolution of ~20 - 30 nm using photoactivated localization microscopy (PALM) (Figure 8).

Practical considerations

Detection
The green and the red fluorescent state of EosFP can be detected with standard filter sets (FITC / GFP filters for the green state or TRITC / DsRed for the red state). Fluorescence of the red state can be detected instantaneously after photoconversion. Green fluorescence can be monitored starting between 6.5 and 12 h after transfection / microinjection of vector / mRNA. Microinjection of purified EosFP allows immediate cell labeling by photoconversion.

Photoconversion
Photoconversion can be achieved by irradiation with light of wavelengths between 350 and 440 nm with a maximal efficiency at ~390 nm. Therefore, standard DAPI filter sets can be used for photoconversion as well as customized filters with maximal transmission at 400 - 440 nm and appropriate lasers, e.g. a 405 nm laser diode. Photoconversion can usually be achieved within a few seconds, depending on the energy output of the light source. However, an increase of the energy beyond a limit set by the maximal conversion rate of EosFP might result in an unwanted bleaching of the red fluorescent state. In such cases, prolonged irradiation with lower light levels should be applied. At present, no negative effects of the photoconversion on expressing cells were reported.

Turnover of the red fluorescent state

Both the green and the red form of EosFP are highly stable at cytosolic pH values. A half-life of ~3 weeks was determined for the red form of wildtype EosFP in coral cells. In developing embryos of Xenopus laevis, the photoconverted stage could be tracked up to 14 days. In dividing cell cultures (HEK293), the red fluorescence could be traced be flow cytometry for up to 9 days.

Cell labeling vs. fusion proteins: Choice of EosFP variants

Two variants of EosFP are available from MoBiTec: The tetrameric wildtype protein (wt-EosFP) and a pseudomonomeric variant in which two copies of an engineered EosFP variant are fused to form a tandem dimer (td-EosFP). Both variants express functionally in a wide range of pro- and eukaryotic cells at a temperature of 37 °C or below. For the labeling of cells or tissues, tetrameric EosFP is the construct of choice. For labeling of subcellular compartements using short oligopeptide signals attached to the marker, both EosFP and td-EosFP can be considered. Although some fusion proteins with tetrameric EosFP are possible, the pseudomonomeric variant td-EosFP is the recommended construct for protein labeling. Fusions to the N-terminus of td-EosFP usually work well. Fusions to the C-terminus are also possible, however, some fusion might fail with proteins requiring a strictly monomeric marker, for instance tubulin.

EosFP-Green to Red Photoconvertible Fluorescent Protein

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pcDNA3-vector, td-EosFP, FLAG®-tagged, lyophilized DNA
MoBiTec
VS-FLP10030 10 ug
626,56 €
pcDNA3-vector, wt-EosFP, FLAG®-tagged, lyophilized DNA
MoBiTec
VS-FLP10020 10 ug
626,56 €
pcDNA3-vector, wt-EosFP, with mitochondrial targeting signal, lyophilized DNA
MoBiTec
VS-FLP10010 10 ug
588,43 €
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