Malek A, Khaledi M G
Department of Chemistry, North Carolina State University, Raleigh, North Carolina, 27695, USA.
Anal Biochem. 1999 Mar 15;268(2):262-9. doi: 10.1006/abio.1998.2975.
The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.
绿色荧光蛋白（GFP）作为一种基因表达报告分子引起了广泛关注。本文研究了毛细管电泳-激光诱导荧光法（CE-LIF）在定量细胞提取物和单细胞中绿色荧光蛋白方面的应用。GFP蛋白的S65T突变体形式在人胚肾（HEK293）细胞中成功表达，并通过荧光显微镜和CE-LIF证实了其产生。突变体S65T的质量检测限为5.3×10⁻²⁰ mol，比野生型GFP的检测限优六倍。由于在低GFP浓度下细胞自发荧光的干扰，通过传统技术如荧光显微镜检测少量GFP是困难的。用产生S65T-GFP的GFP质粒转染HEK293细胞。转染后2小时检测到S65T蛋白的瞬时产生，并在48小时后达到最大值。96小时后蛋白质浓度开始显著下降。对转染GFP质粒后的HEK293细胞进行单细胞分析表明，细胞间S65T-GFP蛋白的产生不均匀。
