pwt-mitoEosFP, with mitochondrial targeting signal BioVector NTCC质粒载体菌种细胞基因保藏中心

pwt-mitoEosFP, with mitochondrial targeting signal

Product
pwt-mitoEosFP vector with tetrameric wildtype EosFP and mitochondrial targeting signal.
Introduction
EosFP was isolated from the stony coral Lobophyllia hemprichii (Wiedenmann, J. et al. 2004). Initially, the
protein matures in a green fluorescent state with an emission maximum at 516 nm. Upon irradiation with violetblue light the chromophore undergoes an irreversible photoconversion to a red state emitting at 581 nm
(Nienhaus, K. et al. 2005a). 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.

Fluorescence properties
Excitation
before photoconversion 506 nm
after photoconversion 571 nm
Emission
before photoconversion 516 nm
after photoconversion 581 nm
Extinction coefficient
before photoconversion 72’000 M-1
cm-1
after photoconversion 41’000 M-1
cm-1
Fluorescence Quantum Yield
before photoconversion 0.70
after photoconversion 0.55
Oligomerization
tetramer
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 (EosFP) (Widenmann, J. et
al. 2004) and a pseudomonomeric variant in which two copies of an engineered EosFP variant are fused to form
a tandem dimer (ptd-EosFP) (Nienhaus, G.U. et al. 2006). Both variants express functionally in a wide range of
pro- and eukaryotic cells at a temperatures 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 ptd-EosFP can be considered. Although some fusion proteins with
tetrameric EosFP are possible, the pseudomonomeric variant ptd-EosFP is the recommended construct for
protein labeling. Fusions to the N-terminus of ptd-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.
pwt-mitoEosFP with tetrameric wildtype EosFP and mitochondrial targeting signal.

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