Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis.
Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis.
复制标题
人胚胎肾细胞中绿色荧光蛋白的毛细管电泳表达和分析。
DOI：
10.1006/abio.1998.2975
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Khaledi,MG
中科院分区：
文献类型：
--
作者：
Malek,A;Khaledi,MG
绿色荧光蛋白（GFP）作为基因表达的报告基因引起了人们的广泛兴趣。本文研究了激光诱导荧光毛细管电泳 (CE-LIF) 在细胞提取物和单细胞中绿色荧光蛋白定量中的应用。 GFP蛋白的S65T突变体在人胚肾（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 的检测质量限为 5.3 × 10−20mol，比野生型 GFP 的检测质量限好六倍。由于低 GFP 浓度下细胞自发荧光的干扰，通过荧光显微镜等传统技术很难检测到少量 GFP。 HEK293 细胞用产生 S65T-GFP 的 GFP 质粒转染。转染后2小时检测到S65T蛋白瞬时产生，并在48小时后达到最大值。 96小时后蛋白质浓度开始显着下降。用 GFP 质粒转染后的 HEK293 细胞的单细胞分析表明，细胞间 S65T-GFP 蛋白的产生不均匀。
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 × 10−20mol, 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.