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EosFP and IrisFP Photoconvertible Fluorescent Protein

VS-FLP10050 - pmIrisFP, Flag-tagged, lyophilized DNA

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IrisFP

Green to Red Photoconvertible and Photoactivatable Fluorescent Protein

Source

mIrisFP is an advanced variant of the green to red photoconvertble protein EosFP that was isolated from the stony coral Lobophyllia hemprichii. mIrisFP differs from monomeric mEosFP by four additional mutations, A69V, K145I, F173S, and Y189A.

Features

  • Photactivatable and photoconvertible 
  • Low tendency for dimer formation
  • Expected localization of fusion proteins shown for various proteins 
  • Suitable for pulse-chase experiments, measurement of Kon and Koff rates, and high resolution microscopy (PALM) 

Description

IrisFP is a photoactivatable fluorescent protein 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. mIrisFP is a monomeric variant. mIrisFP multiple photo-activation modes can be used for pulse-chase experiments combined with subdiffraction-resolution imaging in living cells by using dual-color photo-activation localization microscopy (PALM).
Monomeric mIrisFP has excellent properties as a genetically encoded fluorescence marker. mIrisFP fluorescence can be reversibly switched on and off (useful for high-resolution microscopy, PALM), but also allows for the non-reversible photo-conversion from green to red (useful for dynamic measurements in living cells). The dual photo-activation capability of mIrisFP offers enhanced flexibility and also enables the combination of pulse-chase experiments with super-resolution imaging.

iris1A 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.

Iris2mIrisFP for combined pulse-chase experiments and superresolution imaging. (ad) 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). (eh) 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). (il) 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).

Iris3Schematic view of the light-driven transformations of the mIrisFP chromophore. Suitable wavelengths for photoconversion / photoswitching are given in the arrows

Optical parameters

The chromophores of both the green and red forms are in cis conformation. Excitation of either fluorescent species induces chromophore isomerization to the non-fluorescent trans conformation. The green trans chromophore absorbs maximally at 386 nm with peak extinction coefficient of 12,000 M−1 cm−1, the red trans chromophore absorbs maximally at 446 nm with peak extinction coefficient of 21,000 M−1 cm−1, and isomerize spontaneously to the fluorescent cis form.
mIrisFPs green fluorescent form has a peak extinction coefficient of 47,000 M−1 cm−1 at 486 nm and emits fluorescence with a high quantum yield of 0.54 peaking at 516 nm.
mIrisFPs red fluorescent form has a peak extinction coefficient of 33,000 M−1 cm−1 at 546 nm and emits fluorescence with a high quantum yield of 0.59 peaking at 578 nm.

Iris4Absorption, excitation and emission spectra, depicted by solid, dotted and dashed lines, respectively, were scaled to equal peak amplitudes. Emission spectra of green (red) mIrisFP were taken with excitation at 473 (532) nm; excitation spectra of green (red) IrisFP were measured via the emission at 540 (580) nm. (a) Green mIrisFP and (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, and (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.

Maturation

in vitro: mIrisFP matures with a half-life of 14 min at 37 °C, faster than many engineered fluorescent proteins.
in vivo: in HEK293 cells, mIrisFP green fluorescence appears 15 h after transfection, similar to EGFP (13 h).

Vectors

Cat. #DescriptionAmount
VS-FLP10010pwt-EosFP, with mitochondrial targeting signal, lyophilized DNA10ug
VS-FLP10020pwt-EosFP, FLAG®-tagged, lyophilized DNA10ug
VS-FLP10030ptdEosFP, Flag®-tagged, lyophilized DNA10ug
VS-FLP10040pmEosFP (Thermostab), Flag®-tagged, lyophilized DNA10ug
VS-FLP10050pmIrisFP, Flag®-tagged, lyophilized DNA10ug

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