Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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Vectors for EosFP Photoconvertible Fluorescent Proteins

Strong Tools for Your Fluorescence Microscopy Studies!

Our EosFP Product Line

    MoBiTec offers cloning vectors for constructing protein fusions containing EosFP signal peptide as well as ready-to-use constructs for transfection and subsequent microscopy analysis such as EosFP construct with mitochondrial targeting signal.

Features

  • Diverse EosFP variants
  • Green to red photoconvertible: UV/blue inducible
  • Permanent, bright and fast
  • Photoconvertible and photoactivatable EosFP variant (mIrisFP)

Applications

  • Localization studies of cells, cell compartments and fusion proteins in live cells or tissues
  • Counting and cluster analyses
  • Regional optical marking of cells, cell compartments and EosFP fusion proteins by photoconversion
  • Pulse chase experiments, measurements of kon- and koff rates, and high resolution fluorescence microscopy like PALM

Description

We offer many diverse cloning and expression vectors encoding different variants of EosFP, that can be used for several applications like localization studies of cells, cell compartments and fusion proteins in live cells. All our EosFP variants are photoconvertible, applicable to high resolution fluorescence microscopy like PALM and stable at 37 °C and below. Table 1 gives an overview on all available EosFP variants and their features:

EosFP Vector Description of EosFP Variant
pWT-EosFP,
FLAG®-tagged
Tetrameric wildtype EosFP
ptd-EosFP,
FLAG®-tagged
Pseudomonomeric tandem EosFP
pm-EosFP (Thermostab), FLAG®-taggedOptimized monomeric variant of EosFP for functional expression at 37 °C excellent performance in fusion even with demanding proteins such as tubulin
pmIrisFP,
FLAG®-tagged
Advanced photoconvertible and photoactivatable monomeric EosFP variant that is stable at 37 °C and below
pwt-EosFP,
with mitochondrial targeting signal
Tetrameric wildtype EosFP with mitochondrial targeting signal
ptd-EosFP-Paxillin,
FLAG®-tagged
Pseudomonomeric tandem EosFP fused to Paxillin; ready-to-use construct
ptd-EosFP-BAP31Pseudomonomeric tandem EosFP fused to BAP31; ready-to-use construct
ptd-EosFP-CD8Pseudomonomeric tandem EosFP fused to CD8; ready-to-use construct
Application
Labeling of cells and subcellular compartments to only a limited extend also suitable for protein labeling
Protein labeling, labeling of cells and subcellular compartments
Protein labeling, labeling of cells and subcellular compartments, pulse-chase experiments, measurements of kon- and koff rates
Labeling of mitochondria
Localization studies; suitable as control and adjusting sample for any fluorescence application including high resolution fluorescence microscopy like PALM
Localization studies and counting and cluster analyses, suitable as control and adjusting sample for any fluorescence application including high resolution fluorescence microscopy like PALM
Practical Details
The vectors can be used for direct transfection or as cloning vector for creating signal peptides or protein fusions.
The vector can be used for direct transfection.

Table 1: For more detailed information to each EosFP variant please click on the respective vector name in the table

EosFP - Background

Spectra of the green and red states of EosFP at pH 7 and pH 5.5

Spectra of the green and red states of EosFP at pH 7 and pH 5.5.


  • Solid lines: absorbance
  • Dashed lines: excitation
  • Dotted lines: emission spectra.

(A and C) Green species at pH 7 (A) and pH 5.5 (C). Excitation spectra were measured with emission set to 520 nm. Emission spectra were measured with excitation set to 490 nm. Open circles: conversion yields scaled to the absorbance. Inset in A: In vitro chromophore maturation at 27 °C indicated by the absorbance at 506 nm (solid line, exponential fit).
(B and D) Red species at pH 7 (B) and pH 5.5 (D). Excitation spectra were measured with emission set to 590 nm, emission spectra were measured with excitation set to 560 nm. (Wiedenmann et al., 2004)

Introduction

EosFP was isolated from the stony coral Lobophyllia hemprichii (Wiedenmann et al., 2004). 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 (Nienhaus et al., 2005). The wavelengths required for photoconversion and detection of the green and red fluorescent states can be easily separated. This makes EosFP an excellent choice for localization and co-localization studies and for regional optical marking.

Detection

The green and the red fluorescent state of EosFP can be detected with standard filter sets (FITC and GFP filters for the green state or TRITC and Ds Red for the red state). Fluorescence of the red state can be detected instantaneously after photoconversion. Green fluorescence can be monitored from 6.5 to 12 hours after transfection or microinjection of vector, mRNA etc. 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 achiieved within a few seconds, depending on the energy output of the light source. However, an increse 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. Until now, there has been no negative effects of photoconversion on cells expressing EosFP 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 wild type 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 by flow cytometry for up to 9 days (Leutenegger et al., 2007).

before photoconversion after photoconversion
Excitation 506 nm 569 nm
Emission 516 nm 582 nm
Extinction coefficient 84'000M-1cm-1 33'000M-1cm-1
Fluorescence Quantum Yield 0.66 0.60

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
VS-FLP10050 pmIrisFP, FLAG-tagged (FLAG is a registered trademark of Sigma-Aldrich Co), lyophilized DNA 10 µg 892,00 PDF
VS-FLP10010 pwt-mitoEosFP, with mitochondrial targeting signal, lyophilized DNA 10 µg 537,00 PDF
VS-FLPC1011 ptd-EosFP-Paxillin, FLAG-tagged, (FLAG is a registered trademark of Sigma-Aldrich Co), lyophilized DNA 20 µg 548,00 PDF
VS-FLPC1012 ptd-EosFP-CD8, lyophilized DNA 20 µg 548,00 PDF
VS-FLPC1013 ptd-EosFP-BAP31, lyophilized DNA 20 µg 548,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.