Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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Vectors for EosFP Photoconvertible Fluorescent Proteins

pmIrisFP

Features

  • Expression vector for the fluorescent protein mIrisFP
  • Green to red photoconvertible and photoactivatable
  • Permanent, bright and fast
  • Monomeric- Low tendency for dimer formation
  • Stable at 37 °C and below
  • FLAG®-tagged
  • Suitable for pulse-chase experiments, measurements of kon- and koff- rates, and high resolution fluorescence microscopy like PALM
  • Selective marking of cells, subcellular compartments and fusion proteins by photoconversion
  • Suitable for labeling and tracking of cells, subcellular compartments and fusion proteins:
  • - Ready-to-use construct for cell tracking
  • - Source vector for creating fusions with signal peptides

Description

"pmIrisFP" is a cloning and expression vector, encoding for the photoactivatable fluorescent protein mIrisFP that combines irreversible photoconversion from a green-to a red-emitting form with reversible photo-switching between a fluorescent and a non-fluorescent state in both forms. The protein is an advanced monomeric variant of EosFP that differs from the monomeric mEosFP by four additional mutations: A69V, K145I, F173S and Y189A.
mIrisFP has excellent properties as a genetically encoded fluorescence marker. It matures with a half-life of 14 min at 37 °C, faster than many engineered fluorescent proteins in vitro. In HEK293 cells mIrisFP green fluorescence appears 15h after transfection, similar to EGFP (13h). mIrisFP fluorescence can be switched on and off reversibly (useful for High Resolution Fluorescence Microscopy like PALM), but also allows for the non-reversible photoconversion from green to red (useful for dynamic measurements in living cells). The dual photoactivation capability of mIrisFP offers enhanced flexibilitiy and also enables the combination of pulse-chase experiments with super-resolution imaging. mIrisFP multiple photo-activation modes can also be used for pulse-chase experiments combined with subdiffraction- resolution imaging in living cells by using dual-color Photoactivation Localization Microscopy (dual-color PALM).

Schematic view of the light-driven transformations of the mIrisFP chromophore

Schematic view of the light-driven transformations of the mIrisFP chromophore. Suitable wavelengths for photoconversion / photoswitching are given in the arrows.

Spectroscopic characterization of mIrisFP
    Spectroscopic characterization of mIrisFP
  • Solid lines: absorbance spectra
  • Dotted lines: excitation spectra
  • Dashed lines: emission spectra
  • Absorption, excitation and emission spectra were scaled to equal peak amplitudes.
  • Emission spectra of green mIrisFP were taken with excitation at 473 nm, and emission spectra of red mIrisFP were taken with excitation at 532 nm.
  • Excitation spectra of green mIrisFP were measured via the emission at 540 nm, and excitation spectra of red mIrisFP were taken with emission at 580 nm.
  • (A) Green mIrisFP
  • (B) Green mIrisFP before (green lines) and after (black lines) illumination with 473 nm light.
  • (C) Red mIrisFP (red lines) after photoconversion of green mIrisFP (green lines) with 405 nm light
  • (D) Red mIrisFP before (red lines) and after (black lines) illumination with 532 nm light (corrected for background).
  • All spectra were recorded in sodium phosphate buffer, pH 10.

Optical parameters of mIrisFP:

before photoconversion after photoconversion
Excitation / Emission 485 nm / 516 nm 546 nm / 578 nm
Extinction coefficient 47'000M-1cm-1 33'000M-1cm-1
Fluorescence Quantum Yield 0.54 0.59
Brightness x 1000 25.5 19.3
Switching half-time off / on 8.8 s / 6.4 sa 16.0 s / 4.9 sb
Relaxation half-time 53 min 24 min
Detected photons per burst 176 354

a off-switching: 473 nm light, 30 mW cm-2, on-switching: 405 nm light: 4.5 mW cm-2 b off-switching: 561 nm light, 6 mW cm-2, on-switching: 473 nm light: 0.5 mW cm-2

Applications

    Localization studies of fusion proteins with mIrisFP
A cell expressing paxillin in fusion with mIrisFP

A cell expressing paxillin in fusion with mIrisFP: The insets show the resolution that can be achieved for individual focal adhesions by conventional TIRF microscopy and PALM. Lower row, paxillin dynamics in focal adhesions imaged with super-resolution over a period of 250 seconds.

    Pulse-chase experiments and superresolution imaging with mIrisFP
    mIrisFP for combined pulse-chase experiments and superresolution imaging:
  • (A-D) A PALM image is first acquired using off-on switching of green mIrisFP (A).
  • A subpopulation of mIrisFP molecules in a region of the cell (violet) is photoconverted to the red form (B). These molecules migrate to other parts of the cell (C), which can be observed with PALM using off-on switching of red mIrisFP (D).
  • (E-H) TIRF (E, F) and PALM (G, H) images of a HeLa cell expressing α-actinin-mIrisFP. Closeups of the white boxed areas in E and G are shown in F and H. Fluorescence from green mIrisFP molecules was monitored (90 s total exposure). The violet box in G marks the irradiated region (405 nm laser light, 20 s).
  • (I–L) PALM images detecting red-converted mIrisFP over the intervals 0–37.5 s (I, J) and 100–300 s (K, L) after the end of photoconversion. Closeups of boxed areas in I and K are shown in J and L. Scale bars, 10 µm (E, G, I, K) and 1 µm (F, H, J, L).

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
VS-FLP10050 pmIrisFP, FLAG-tagged (FLAG is a registered trademark of Sigma-Aldrich Co), lyophilized DNA 10 µg 892,00 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 pmIrisFP, FLAG®-tagged (pic)

Sequence of pmIrisFP, FLAG®-tagged (txt)

Literature

  1. Fuchs, J., Boehme, S., Oswald, F., Hedde, P.N., Krause, M, Wiedenmann, J., and Nienhaus, G.U. (2010). Imaging Protein Movements in Live Cells with Super-resolution Using mIrisFP. Nature Methods 7, 627 – 630.
  2. McKinney, S.A., Murphy, C.S., Hazelwood, K.L., Davidson, M.W. & Looger, L.L. (2009). A bright and photostable photoconvertible fluorescent protein. Nature Methods 6, 131-133.