Robić Goran, Lacorte Cristiano, Miranda Everson A
Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, CEP 13083-970 Campinas, SP, Brazil.
Anal Biochem. 2009 Sep 1;392(1):8-11. doi: 10.1016/j.ab.2009.05.016. Epub 2009 May 18.
The main use of green fluorescent protein (GFP) is as a reporter system, where the existence of the protein is usually determined visually using fluorescent microscopy. Although fluorescence-based quantification of GFP is possible, background fluorescence in plants and in plant extracts was observed by our group. Another phenomenon we observed that makes quantification difficult is the increased level of GFP fluorescence in Nicotiana benthamiana leaf extracts, probably the result of dimerization of GFP molecules promoted by interaction with some component(s) of tobacco extracts. In the current work, the background fluorescence was minimized and the enhancement of GFP fluorescence in tobacco extracts was eliminated with the addition of urea to the measured solution so that a simple quantification assay for the GFP in the tobacco extracts could be established.
绿色荧光蛋白（GFP）的主要用途是作为一种报告系统，通常通过荧光显微镜在视觉上确定该蛋白的存在。虽然基于荧光对GFP进行定量是可行的，但我们团队观察到植物及其提取物中存在背景荧光。我们观察到的另一个使定量变得困难的现象是，本氏烟草叶片提取物中GFP荧光水平增加，这可能是由于与烟草提取物的某些成分相互作用促进了GFP分子二聚化的结果。在当前的工作中，通过向测量溶液中添加尿素，将背景荧光降至最低，并消除了烟草提取物中GFP荧光的增强，从而可以建立一种针对烟草提取物中GFP的简单定量测定方法。
