title: Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials.
abstract: Green fluorescent proteins (GFPs) are widely used in tracing transgene expression and have been known as convenient and efficient markers for plant transformation. However, sometimes researchers are still puzzled by the weak fluorescence since it makes the observation of GFP signals and confirmation of transgenic plants difficult. In this investigation, we explored the possibility of enhancing the weak signals by changing the pH environment of detection and took microplate reader as a more effective instrument compared to traditional fluorescent microscope to detect the weak signals. It was found that the fluorescence intensity of enhanced GFP (EGFP) in transgenic plants can be increased 2-6 folds by altering the environmental pH, and the concentration of EGFP at a large scale (ranged from 20 ng/ml to 20 μg/ml) can be detected and quantified. It can exclude the influence of degradation fragment and hence facilitate later analysis; these advantages were further verified by comparing with western blotting and confocal microscopy. It was reliable and effective for the qualitative and quantitative analysis of transgenic plants and was more suitable for the detection of very weak fluorescent signals.
authors.affiliation: Key Laboratory of Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan, China. wujj8108@gmail.com
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Wu
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Liu
authors.forename: Meng-Xiang
authors.initials: MX
authors.lastname: Sun
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal / Q000379:methods; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins