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We used Zeiss imaging software to concatenate the pre-bleach and post-bleach image sets and generated pseudo-colored images showing FRET efficiency values throughout each cell; all images were background-subtracted.
(B ) Single molecule time traces showing FRET for an immobilized ssDNA with two identical 5 nt homology sequences at HS1 and HS2 in a poly T sequence background.
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Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene (TPhenBDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, PerU or OxoPyU or PerU, forming two stable chimeric DNA duplexes.
Table S1 shows FRET values for individual cells using the three quantitative FRET methods.
The rest of the proteins showed FRET ratio values +/− 20% of the 1b genotype (Figure 5C).
Fusion of fluorescent proteins to the carboxy-termini of α and β subunits show FRET efficiencies of 15% 20% corresponding to a distance of 62 Å–66 Å (Table 1).
The βPS-GFP/talinIBS2-mCherry pair did not show FRET in muscles, but showed substantial FRET in wing adhesions.
At this threshold, a large fraction of detected species showed FRET: 0.66 (FD), 0.42 (LA).
The intermediate decay time can be attributed to bigger oligomers that show FRET from the AF488 to AF647-labeled subunits.
Furthermore, ∼30% of the unwinding traces with 100 μM dTTP showed FRET fluctuations that appeared nonmonotonic, indicating partial reannealing of DNA and backward movements by the T7 helicase.
The scatter plot demonstrated that TMRM intensity is significantly reduced in all cells that showed FRET ratio change from the control.
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