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Aequorea green fluorescent protein analysis by flow cytometry
J D Ropp1, C J Donahue, D Wolfgang-Kimball
1Department of Pulmonary Research, Genentech Inc., South San Francisco, California, USA.
Cytometry
|December 1, 1995
Summary
We compared a green fluorescent protein (GFP) mutant (S65T-GFP) to wild-type GFP (wt-GFP) for gene expression sensitivity. S65T-GFP showed higher fluorescence with 488 nm excitation, while wt-GFP was brighter at 407 nm.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Green fluorescent protein (GFP) from Aequorea victoria is a valuable reporter for gene expression.
- Wild-type GFP (wt-GFP) has a bimodal absorption spectrum with peaks at 395 nm and 470 nm.
- A GFP mutant (S65T-GFP) was engineered with a single absorption peak at 490 nm to enhance sensitivity.
Purpose of the Study:
- To compare the fluorescence sensitivity of S65T-GFP and wt-GFP.
- To determine optimal excitation wavelengths for each GFP variant.
- To assess the utility of these GFPs with standard laser excitation sources.
Main Methods:
- Transient transfection of 293 cells with S65T-GFP and wt-GFP.
- Flow cytometry analysis of fluorescence intensity using argon ion (488 nm, UV) and krypton ion (407 nm) lasers.
- Spectrofluorometric analysis of emission spectra.
- Investigation of cellular autofluorescence at different wavelengths.
Main Results:
- S65T-GFP exhibited the highest fluorescence signal when excited at 488 nm.
- wt-GFP showed significantly higher fluorescence intensity at 407 nm compared to 488 nm or UV excitation.
- Cellular autofluorescence was notably lower at 407 nm than at UV or 488 nm.
Conclusions:
- The choice of GFP variant and excitation wavelength impacts fluorescence sensitivity.
- S65T-GFP offers enhanced sensitivity with 488 nm excitation, suitable for standard filter sets.
- wt-GFP provides higher signal-to-noise ratios at 407 nm due to reduced autofluorescence, making it advantageous for specific applications.