Figure 1.

Fluorescence properties of selected green fluorescence proteins. Different fluorescence proteins (color coding and numbering are consistent throughout the whole figure) were expressed in yeast as an endogenously expressed C-terminally tagged Ypd1 fusion protein. (a) Fluorescence lifetime histograms normalized to the maximum of each curve are shown. (b) Raw fluorescence intensity traces that were measured for 45 s are shown. Presented data in (a) and (b) correspond to average traces from five measurements. (c) Immunoblot and detection of the Ypd1-GFP fusion proteins using anti-GFP antibodies are shown. Please note that this does not allow direct comparison of protein levels because different GFP variants may cover different range of epitopes recognized by the polyclonal anti-GFP antibodies. No degradation products were detected, indicating that no free GFP is present inside the cells (62). (d) Absolute protein concentrations as determined by FCS are shown. (e) Fluorescence molecular brightness normalized to the mean of Ypd1-sfGFP. Data in (d) and (e) are the result of five measurements each. Boxes in the boxplot correspond to the first and third quartiles, the horizontal line represents the median. The whiskers show first quartile − 1.5 × interquartile range and third quartile + 1.5 × interquartile range, respectively (boxplot description is consistent throughout the whole manuscript).