Acquisition and Stability of GFP Fluorescence 1 1 31 Photobleaching

The GFP fluorescence is very stable in a fluorometer (Ward, W. W., personal communication). Even under the higher intensity illumination of a fluorescence microscope, GFP is more resistant to photobleaching than is fluorescein (25). The fluorescence of wt GFP, GFP-S65T, and RSGFP is quite stable when illuminated with 450-490 nm light (the major excitation peak for GFP-S65T and RSGFP, but the minor peak for wt GFP). Some photobleaching occurs when wt GFP is illuminated near its major excitation peak with 340390 nm or 395^140 nm light (4). The rate of photobleaching is less with lower energy lamps such as QTH or mercury lamps. High-energy xenon lamps should be avoided, as these may cause rapid photodestruction of the GFP chro-mophore. The rate of photobleaching varies with the organism being studied; GFP fluorescence is quite stable in Drosophila (25) and zebrafish. In C elegans, 10 mMNaN3 accelerates photobleaching (4).

1 1 3.2 Stability to Oxidation/Reduction

The GFP needs to be m an oxidized state to fluoresce; strong reducing agents, such as 5 mM Na2S204 or 2 mM FeS04, convert GFP into a non-fluorescent form, but fluorescence is fully recovered after exposure to atmospheric oxygen (26). Weaker reducing agents, such as 2% P-mercaptoethanol, 10 mM dithiothreitol (DTT), 10 mM reduced glutathione, or 10 mML-cys-teine, do not affect the fluorescence of GFP (26). GFP fluorescence is not affected by moderate oxidizing agents (Ward, personal communication).

1 1 3.3 Stability to Chemical Reagents

The GFP fluorescence is retained in mild dénaturants, such as 1% SDS or 8M urea, after fixation with glutaraldehyde or formaldehyde, but fully denatured GFP is not fluorescent. The GFP is very sensitive to some nail polishes used to seal coverslips (4,25); therefore, use molten agarose or rubber cement to seal coverslips on microscope slides. Fluorescence is also quenched by the nematode anesthetic phenoxypropanol (4). The GFP fluorescence is irreversibly destroyed by 1% H202 and sulfhydryl reagents such as 1 mMDTNB (5,5'-dithio-bis-[2-nitrobenzoic acid]) (26). Fluorescence survives pH 7-12, but intensity decreases at pH 5.5—7 0 (27). Many organic solvents can be used at moderate concentrations without abolishing fluorescence; however, the absorption maximum may shift (28).

GFP dimerizes via hydrophobic interactions at protein concentrations above 5—10 mg/mL and high salt concentrations with a fourfold reduction in the absorption at 470 nm (Ward, personal communication). Dimer formation is not required for fluorescence, and monomeric GFP is the form of the reporter expressed in most model systems. Protein Stability: In Vitro

GFP is exceptionally resistant to heat (Tm = 70°C), alkaline pH, detergents, chaotropic salts, organic solvents, and most common proteases, except pronase (27—30). Some GFP fluorescence can be observed when nanogram amounts of protein are resolved on native or 1% SDS polyacrylamide gels (2).

Fluorescence is lost if GFP is denatured by high temperature, extremes of pH, or guamdinium chloride but can be partially recovered if the protein is allowed to renature (27,31). A thiol compound may be necessary to renature the protein into the fluorescent form (32).

1.1 3.5 Protein Stability: In Vivo

The GFP appears to be stable when expressed in various organisms. However, no measurement of the half-life of GFP has been reported Temperature Sensitivity of GFP Chromophobe Formation

Although fluorescent GFP is highly thermostable (30), it appears that the formation of the GFP chromophore is temperature sensitive. In yeast, GFP fluorescence was strongest when the cells were grown at 15°C, decreasing to about 25% of this value as the incubation temperature was raised to 37°C (33). However, GFP and GFP-fusions synthesized in S. cerevisiae, at 23 °C retain fluorescence despite a later shift to 35°C (33). It has also been noted that E. coli expressing GFP show stronger fluorescence when grown at 24°C or 30°C as compared to 37°C (19] Ward, personal communication). Mammalian cells expressing GFP have also been seen to exhibit stronger fluorescence when grown at 33°C as compared to 37°C (11).

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