Quantitative Comparison of Monomeric StayGold Variants Using Protein Nanocages in Living Cells
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Abstract
To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.
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RepresentativeEGFP and mStayGold nanocage photobleaching images. Images are scaled to the same absolute intensities
these are not the same nanocages being imaged at the three different timepoints, correct? would it be possible to image the same immobilized nanocages over time?
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we present a new method to compare fluorescent proteins on a molecule-by-molecule basis inphysiological conditions independent of relative expression levels or ratio measurements to other fluorescentproteins
As a tool to evaluate FPs in the future, what does this approach provide above what can be learned with absorbance/spectroscopy measurements performed on purified FPs?
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omparable peripheral cellregions of D-Mannitol-treated RPE cells expressing the indicated FP nanocage fusions
In the last panel (E138D), it looks like there are two dim nanocages and the rest are brighter. What could be the source of the variation?
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Thus, each nanocage carries sixty FPs (Fig. 1b) allowing comparison of the absolute fluorescence intensitybetween individual nanocages with different FPs
this is a very clever way to benchmark FPs that ensures a direct head-to-head performance comparison. By making comparisons between individual nanocages, this technique largely avoids confounds introduced by variations in expression level and cell-to-cell variability.
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