Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: Example of practical brightness quantification using the T2A peptide linker. Cells expressing either yoeCFP-T2A-mCherry or mTq2-T2A-mCherry were grown to midlog and visualized using a widefield microscope. yoeCFP shows a low brightness compared to mCherry. In contrast, mTq2 shows a higher brightness than mCherry. Calibration bar indicates the ratio value when dividing the CFP by the RFP channel (i.e. the relative brightness to mCherry).

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: Expressing, Microscopy

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: In vivo brightness and photostability of FPs. A) In vivo brightness of FPs measured by normalizing the fluorescence intensity of single-cells expressing FP-T2A-FP and dividing the fluorescence of the FP of interest to the control FP (either mTq2 or mCherry). B) Photostability of FPs. Per FP, a time-lapse movie was recorded and the photostability was measured as the fluorescent fraction of the last time frame compared to the first frame. Dots represent relative brightness or photostability of an individual cell, boxes indicate median with quartiles, whiskers indicate the 0.05–0.95 fraction of the datapoints. C) Overview of the brightness and photostability of all characterised FPs. D) Coefficient of variation (CV) of the mean brightness of 3 days as an indication of day-to-day variation.

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: In Vivo, Fluorescence, Expressing

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: pH sensitivity of FPs. A) Yeast cells were incubated for 2 hours in citric-acid/Na 2 HPO 4 buffers set at pH 3-8 with 2 mM 2,4-DNP and fluorescence was measured using a fluorescent plate reader. Per FP, at least 3 technical replicates were measured. Afterwards, fluorescence was normalized to the pH giving the highest fluorescence and a Hillfit was performed to determine the hill coefficient and pKa value, plotted at the y- and x-axis, respectively. B) mTq2 is an example of an FP that shows different pH sensitivity. pH calibration in vitro was performed using purified proteins in a Citric Acid – Sodium Citrate buffer (pH 3 – 5.4) and a NaH 2 PO 4 /Na 2 HPO 4 0.1 M buffer (pH 5.9-8). Dots represent mean of at least 3 replicates, error bars indicate SD. C) pH curve of sYFP2 which shows an offset (fluorescence plateau) at low pH. This offset gives different values of the pKa (red point, which is the pH that gives a 50% decrease between 1 and the offset) and the pH 50% which gives an absolute 50% decrease in fluorescence (blue point). Dots represent mean of at least 3 replicates, error bars indicate SD.

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: Incubation, Fluorescence, In Vitro, Purification