A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

Remans, T., Schenk, P. M., Manners, J. M., Grof, C. P. L. and Elliott, A. R. (1999) A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants. Plant Molecular Biology Reporter, 17 4: 385-395.


Author Remans, T.
Schenk, P. M.
Manners, J. M.
Grof, C. P. L.
Elliott, A. R.
Title A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants
Journal name Plant Molecular Biology Reporter   Check publisher's open access policy
ISSN 0735-9640
Publication date 1999-12
Sub-type Article (original research)
DOI 10.1023/A:1007654318401
Volume 17
Issue 4
Start page 385
End page 395
Total pages 11
Place of publication Heidelberg
Publisher Springer
Language eng
Abstract The Green Fluorescent Protein (GFP) from Aequorea victor-in has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER Variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +/- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 mu g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 mu g and 2.11 mu g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfPS-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 mu g mGFP5-ER per mg extractable protein.
Keyword Biochemical Research Methods
Plant Sciences
Green Fluorescent Protein
Sugarcane
Tobacco
Transgene Expression Level
Green Fluorescent Protein
Expression
Cells
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Biological Sciences Publications
 
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Created: Mon, 13 Aug 2007, 11:45:25 EST