Abstract
Green fluorescent protein (GFP) is a popular qualitative reporter protein used to study different aspects of plant biology. However, to be used as a reliable quantitative reporter in expression studies using fluorescence based assays, methods to eliminate interfering endogenous molecules must be considered. Therefore, a standard curve based solid phase fluorescent immunoassay that eliminates the effects of interfering endogenous molecules was developed to quantify the GFP levels in soluble green extracts prepared from plants. Microtiter plates coated with anti-GFP were used to capture GFP from soluble plant extracts, interfering endogenous molecules was eliminated by washing without disturbing the anti-GFP binding of GFP, and then the fluorescence intensity of bound GFP was measured using a spectrofluorometer. We report in this study the use of this method to quantify the expression levels of soluble modified GFP in transgenic Arabidopsis thaliana.
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Abbreviations
- CaMV:
-
cauliflower mosaic virus
- smGFP:
-
soluble modified green fluorescent protein
- RFUs:
-
relative fluorescence units
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Acknowledgments
We thank Dr. Phil Mullineaux and Dr. Roger Hellens at John Innes Centre for providing the pGII0029 vector and the Nottingham Arabidopsis Stock Centre (NASC) for providing the 35S-smGFP clone. We thank Johan R. Lillehaug, Department of Molecular Biology, University of Bergen, for helpful discussions. This work was supported by grants from the Hedmark County in Norway.
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Johansen, W., Stenseth, EB. & Wilson, R.C. A Rapid and Sensitive Fluorimetric Protocol for the Quantification of Green Fluorescent Protein in Soluble Protein Extracts from Transgenic Arabidopsis . Plant Mol Biol Rep 25, 45–54 (2007). https://doi.org/10.1007/s11105-007-0006-3
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DOI: https://doi.org/10.1007/s11105-007-0006-3