Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

Products

EosFP - Product Line

pwt-EosFP (Order no: VS-FLP10020)

Features

  • Expression and cloning vector for fluorescent tetrameric wt-EosFP
  • Green to red photoconvertible
  • Permanent, bright and fast
  • Stable at 37 °C and below
  • FLAG®-tagged
  • Selective marking of cells and subcellular compartments by photoconversion
  • Applicable to High Resolution Fluorescence Microscopy like PALM
  • Suitable for tracking of cells and subcellular compartments:
  1. Ready-to-use construct for cell tracking
  2. Source vector for creating fusions with signal peptides

Description

"pwt-EosFP" is a cloning and expression vector encoding for the fluorescent green-to-red photoconvertible wildtype EosFP protein (wt-EosFP). The wildtype protein builds a tetrameric quaternary structure (Nienhaus et al., 2005). It is functionally expressed in a wide range of pro- and eukaryotic cells at a temperature of 37 °C and below. wt-EosFP is particularly suitable as localization marker of cells (see Fig. 1), tissue and subcellular compartments. The construct pwt-EosFP can be used directly in transfection and subsequent analyses by fluorescence microscopy. For labeling of subcellular compartments, short oligopeptide signals can be fused to the marker protein. The cloning site is shown in the vector map. Although some fusion proteins with tetrameric wt-EosFP are functional, we recommend using td-EosFP, mEosFP (thermostable) or mIris fusions. The photoconversion feature makes wt-EosFP applicable to high resolution fluorescence microscopy as well as it enables selective marking of cells and subcellular compartments.

Applications

    Tracking of cells (cell fate mapping, tracking of metastases) To use "pwt-EosFP" for cell labeling, the expression vector is introduced into the cell by transfection of DNA or by microinjection of in vitro transcribed mRNA. Alternatively purified recombinant protein can be microinjected. As shown in Fig. 2, the desired cell or cell group can be labeled and tracked by photoconversion.
Tracking of cells in an embryo of Xenopus laevis

Fig. 1 Tracking of cells in an embryo of Xenopus laevis. Purified wt-EosFP was microinjected at stage 2. After photoconversion of a single blastomer, the fate of descendent cells can be followed by the red fluorescence. (Picture modified from Wacker et al., 2007).

ptd-EosFP (Order no: VS-FLP10030)

Features

  • Expression vector for fluorescent EosFP tandemdimer (td-EosFP)
  • Green to red photoconvertible
  • Permanent, bright and fast
  • Stable at 37 °C and below
  • FLAG®-tagged
  • Selective marking of selected mitochondria by photoconversion
  • Applicable to high resolution fluorescence microscopy like PALM
  • Suitable for tracking of cells and subcellular compartments:
  • - Ready-to-use construct for cell tracking
  • - Source vector for creating fusions with signal peptides

Description

"ptd-EosFP" is a cloning and expression vector encoding for the fluorescent green-to-red photoconvertible EosFP tandem dimer (td-EosFP), a pseudomonomeric variant in which two copies of an engineered EosFP variant are fused to form a functional monomer (Nienhaus, G.U. et al. 2006). td-EosFP is particularly sutiable as localization marker of cells and for tracking of subcellular compartments (See Fig. 3) and fusion proteins. The protein is functionally expressed in a wide range of pro- and eukaryotic cells at a temperature of 37 °C and below. For cell tracking, the vector construct can be used directly for transfection or microinjection and subsequent analyses by fluorescence microscopy. For creating signal peptide or protein fusions, the appropriate signal sequence or the desired gene has to be cloned into the provided cloning site of the "ptd-EosFP" vector. The photoconversion feature makes ptd-EosFP applicable to high resolution fluorescence microscopy and enables selective marking of cells, subcellular compartments and fusion proteins.

Applications

    Tracking of subcellular compartments td-EosFP can be fused to a subcellular targeting signal. td-EosFP fused to the targeting signal of mitochondria is shown in Fig. 2 The organelle or parts thereof can be labeled by photoconversion and their movements can be monitored subsequently.
    Localization Studies of td-EosFP Fusions by Fluorescence Nanoscopy Fig. 3 shows EosFP fused to a protein of interest. The subcellular localization can be determined using Photoactivated Localization Microscopy (PALM) with a resolution of ~20 - 30 nm. (Betzing et al., 2006)
Labeling of cellular compartments

Fig. 2 Labeling of cellular compartments. td-EosFP was targeted to mitochondria. After photoconversion of a single mitochondrion, fusion and fission events can be tracked. (Picture courtesy of Michael Davidson, modified from Wiedenmann & Nienhaus, 2006.).

 Application of td-EosFP in fluorescence nanoscopy

Fig. 3 Application of td-EosFP in fluorescence nanoscopy. td-EosFP was fused to vinculin and expressed in gray fox lung fibroblast cells. Using Photoactivated Localization Microscopy (PALM) the localization in focal adhesions can be imaged with a resolution of ~20 - 30 nm. The figure shows a normal widefield image of a cell. The inset shows a PALM image of parts of a single focal adhesion indicated by the white rectangle. Arrows indicate a partial network structure. (Picture courtesy of Michael Davidson, modified from Shaner et al., 2007).

pmEosFP (Order no: VS-FLP10040)

Features

  • Expression vector for fluorescent monomeric EosFP (mEosFP, Thermostab) protein
  • Optimized monomeric variant for functional expression at 37 °C
  • Excellent performance in fusion even with demanding proteins
  • Green to red photoconvertible
  • Permanent, bright and fast
  • FLAG®-tagged
  • Selective marking of selected mitochondria by photoconversion
  • Applicable to high resolution fluorescence microscopy like PALM
  • Suitable for tracking of cells, subcellular compartments and fusion proteins:
  • - Ready-to-use construct for cell tracking
  • - Source vector for creating fusions with signal peptides

Description

"pmEosFP (Thermostab)" is a cloning and expression vector encoding for an advanced variant of the monomeric fluorescent green-to-red photoconvertible EosFP (mEosFP). The marker was optimized for functional expression at 37 °C, especially in fusion with other proteins. mEosFP (Thermostab) retains the monomeric nature of its predecessor and shows an excellent performance in fusion even with demanding proteins such as tubulin (See Fig. 5). mEosFP (Thermostab) can be efficiently converted from green to red by a light pulse at wavelengths between 360 and 430 nm. For cell tracking, the vector construct can be used directly for transfection or microinjection and subsequent analyses by fluorescence microscopy.
For creating signal peptide or protein fusions, the appropriate signal sequence or the desired gene has to be cloned into the provided cloning site of the "pmEosFP (Thermostab)" vector. The photoconversion feature makes mEosFP (Thermostab) applicable to high resolution fluorescence microscopy and enables selective marking of cells, subcellular compartments and proteins.

Applications

    Localization studies of mEosFP fusion proteins and cell tracking:
    mEosFP (Thermostab) was used for detecting ribosomal binding protein (RBP) and tubulin within cells. Both proteins showed nuclear localization (See Fig. 4-A). A selected fraction of those proteins were tracked by photoconversion from green to red by a light pulse between 360 and 430 nm. (See Fig. 4-B: RBP-mEosFP (Thermostab); C: tubulin-mEosFP (Thermostab)). The marker protein can also be used to study cell movement, e.g. during embryonic development. Here mEosFP (Thermostab) was used for cell tracking in a Xenopus embryo.
Localization studies with mEosFP (Thermostab)

Fig. 4 Localization studies with mEosFP (Thermostab) (A) mEosFP (Thermostab) fused to tubulin.
Conversion from green to red with a light pulse between 360 and 430 nm is shown from B to D. (B) Nuclear localization of RBP-mEosFP (Thermostab) (C) Nuclear localization of tubulin-mEosFP (Thermostab) (D) mEosFP (Thermostab) used for cell tracking in a Xenopus embryo.

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
VS-FLP10020 pwt-EosFP, FLAG-tagged (FLAG is a registered trademark of Sigma-Aldrich Co), lyophilized DNA 10 µg 572,00 PDF
VS-FLP10030 ptd-EosFP, FLAG-tagged (FLAG is a registered trademark of Sigma-Aldrich Co), lyophilized DNA 10 µg 572,00 PDF
VS-FLP10040 pmEosFP(Thermostab), FLAG-tagged (FLAG is a registered trademark of Sigma-Aldrich Co), lyophilized DNA 10 µg 679,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

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Vector maps and Sequences

Vector map of pwt-EosFP, FLAG®-tagged (pic)
Vector map of ptd-EosFP, FLAG®-tagged (pic)
Vector map of pmEosFP(Thermostab), FLAG®-tagged (pic)

Sequence of pwt-EosFP, FLAG®-tagged (txt)
Sequence of ptd-EosFP, FLAG®-tagged (txt)
Sequence of pmEosFP(Thermostab), FLAG®-tagged (txt)

Literature

  1. Wiedenmann, J., Oswald, F., Heilker, R., & Nienhaus, G. U. (2007) Fluoreszente Proteine aus den Ozeanen - Neue Werkzeuge für die zelluläre Bildgebung. BIOforum 30/5, 20-22.
  2. Shaner, N.C, Patterson, G.H. and Davidson, M.W. (2007). Advances in fluorescent protein technology. J Cell Sci 120, 4247-4260. Pubmed
  3. Leutenegger, A., D’Angelo, C., Matz, M.V., Denzel, A., Oswald, F., Salih, A., Nienhaus, G.U. & Wiedenmann, J. (2007). It’s cheap to be colorful: Anthozoans show a slow turnover of GFP-like proteins. FEBS Journal, 274, 2496–2505. Pubmed
  4. Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642-1645. Pubmed
  5. Nienhaus, G.U., Nienhaus, K., Hölzle, A., Ivanchenko, S., Röcker, C., Renzi, F., Oswald, F., Wolff, M., Schmitt, F., Vallone, B., Weidemann, W., Heilker, R., Nar, H., & Wiedenmann, J. (2006). Photoconvertible Fluorescent Protein EosFP - Biophysical Properties and Cell Biology Applications. Photochem. Photobiol. 82, 351-358. Pubmed
  6. Wacker, S., Oswald, F., Wiedenmann, J., & Knöchel, W. (2007). A green to red photoconvertible protein as analyzing tool for early vertebrate development. Dev. Dyn., 236, 473-480. Pubmed
  7. Wiedenmann, J. & Nienhaus, G.U. (2006). Live-cell imaging with EosFP and other Photoactivatable Marker Proteins of the GFP Family. Expert Rev. Proteomics 3, 361-374. Pubmed
  8. Nienhaus, K., Nienhaus, G. U., Wiedenmann, J., & Nar, H. (2005). Structural Basis for Photo-Induced Protein Cleavage and Green-to-Red Conversion of Fluorescent Protein EosFP. Proc. Natl. Acad. Sci. U.S.A. 102, 9156-9159. Pubmed
  9. Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Röcker, C., Salih, A., Spindler, K.D., & Nienhaus, G.U. (2004). EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):15905-10 Pubmed