DsRed surprisingly takes days at room temperature to reach full red fluorescence. At room temperature, a sample of purified protein initially shows a major component of green fluorescence (ex/em 475/499 nm), which peaks in intensity around 7 h and decreases to nearly zero over 2 days. Meanwhile the red fluorescence reaches half its maximal fluorescence after approximately 27 h and requires >48 h to reach >90% of maximal fluorescence.
Fully matured DsRed in our hands has an extinction coefficient of 75,000 and a fluorescence quantum yield of 0.7, much higher than the values of 22,500 and 0.23 previously reported. We have no explanation for the difference except that the lower values might have been measured on incompletely matured protein.
Screening of random mutants produced mutants that appeared green or yellow and were caused by substitutions K83E, K83R, S197T, and Y120H. K83R had the lowest percentage conversion to red and proved very useful as a stable version of the immature green-fluorescing form of DsRed