Oxyradical-induced GFP damage and loss of fluorescence

Denaturation studies reveal significant differences between GFP and blue fluorescent protein

“…We and others have previously shown that denaturation of GFP using heat or other agents was surprisingly very much pH dependent [15,18,16]. This effect of pH on structural stability of GFP is further supported by the crystallographic observations by Jain and Ranganathan who reported minor but significant structural changes in GFP between pH values of 8.5 and pH 5.5 [17].…”

“…This hypothesis is further supported by the crystallographic observations by Jain and Ranganathan that there are minor but significant structural changes in GFP between pH values of 8.5 and pH 5.5 [17]. Additionally, we have recently reported that oxyradicals can be used to denature GFP and that this oxyradical-dependent loss of GFP fluorescence is also pH dependent, such that GFP is most labile to oxy-radical induced damage at pH 6.5 as compared to pH 7.5 or pH 8.5 [18].…”

“…Recently it has been shown that the GFP fluorescence is pH dependent, a property that has been exploited to use GFP as intracellular pH indicators [5,10]. Additionally, we have shown that GFP could also be used to develop a sensitive and cheap assay to screen antioxidants [18]. One of the other interesting properties of GFP is its unusual stability to heat, pH, proteases, and denaturants, which is probably due to the tight and compact "␤-can" structure of the GFP molecule [11][12][13][14].…”

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging

“…The first is the active tissue decoloring ability. This method is milder for proteins, as opposed to the simple flushing or harsh decolorization methods with peroxidase or acetone used previously 30,31 . This enables a wide range of applications for not only the brain but also other organs inside ( Table 1).…”

Ultramicroscopy: development and outlook

“…These peroxide concentrations clearly diminish the success of chemical clearing since they quench the fluorescence of GFP. 31 Also, benzyl alcohol and benzyl benzoate used for the preparation of BABB are already often contaminated with peroxides when delivered by the manufacturer. Thus, it is necessary to eliminate peroxides by means of solvent extraction or column chromatography and to then store the chemicals under argon to prevent them from reacting with oxygen.…”