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Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus

Correlation between EGFP concentration and red fluorescence intensity following irradiation.EGFP was diluted in PBS buffer at the following concentrations: 0.875, 1.7, 3.4, 6.8, 13.75, 27.5, 55 μM. All photoconversion and imaging parameters were kept similar for all tested EGFP concentrations. Data are from 3 independent experiments.
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f3: Correlation between EGFP concentration and red fluorescence intensity following irradiation.EGFP was diluted in PBS buffer at the following concentrations: 0.875, 1.7, 3.4, 6.8, 13.75, 27.5, 55 μM. All photoconversion and imaging parameters were kept similar for all tested EGFP concentrations. Data are from 3 independent experiments.

Mentions: GFP will be present in a variety of concentrations in transgenic cells and organisms, depending on levels of protein expression and stability. We investigated the intensity of the fluorescent signal relative to concentration of purified GFP. Sufficient GFP must be present to observe the photoconverted form, and the signal appears to saturate at approximately 30 μM (Fig. 3).


Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

Correlation between EGFP concentration and red fluorescence intensity following irradiation.EGFP was diluted in PBS buffer at the following concentrations: 0.875, 1.7, 3.4, 6.8, 13.75, 27.5, 55 μM. All photoconversion and imaging parameters were kept similar for all tested EGFP concentrations. Data are from 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493561&req=5

f3: Correlation between EGFP concentration and red fluorescence intensity following irradiation.EGFP was diluted in PBS buffer at the following concentrations: 0.875, 1.7, 3.4, 6.8, 13.75, 27.5, 55 μM. All photoconversion and imaging parameters were kept similar for all tested EGFP concentrations. Data are from 3 independent experiments.
Mentions: GFP will be present in a variety of concentrations in transgenic cells and organisms, depending on levels of protein expression and stability. We investigated the intensity of the fluorescent signal relative to concentration of purified GFP. Sufficient GFP must be present to observe the photoconverted form, and the signal appears to saturate at approximately 30 μM (Fig. 3).

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus