一种简便的高通量细胞内GFP报告基因检测方法  

A simple and high throughput screening method for GFP reporter assay in cells

作　　者：杨少哲[1,2] 石海军[1,2] 褚欣然[1,2] 周小玲[1,2] 孙平楠[1,2] 

机构地区：[1]汕头大学医学院干细胞P2实验室,广东汕头515041 [2]广东省感染病与分子免疫病理重点实验室,广东汕头515041

出　　处：《癌变．畸变．突变》2015年第5期394-398,403,共6页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81571994;81570567);教育部留学回国人员科研启动基金(第49批);广东省自然科学基金(2014A030313483;2015A030313447);汕头大学医学院李嘉诚基金

摘　　要：目的:建立一种简单、快速、高通量的检测GFP蛋白的方法。方法:用TECAN多功能酶标仪Infinite M200pro检测GFP蛋白在不同缓冲液中的荧光强度;用酶标仪检测含GFP的蛋白样品梯度稀释后的荧光强度,建立荧光强度与GFP相对含量之间的标准曲线;分别比较荧光酶标仪的检测结果与流式细胞术和Western-blotting检测结果的相关性。结果:使用50 mmol/L TrisHCl(p H=10)的缓冲液稀释GFP蛋白更有利于检测低浓度的GFP;荧光酶标仪检测GFP的荧光读数同GFP相对含量之间的回归系数2R>0.99。荧光酶标仪检测GFP蛋白的结果与流式细胞术、Western-blotting的检测结果之间均呈现明显的相关性。结论:建立的利用多功能酶标仪检测GFP蛋白方法方便快捷,可以实现短时间对大样本进行检测。OBJECTIVE： To establish a simple, fast and high throughput screening method for green fluorescent protein （GFP） assay. METHODS： We applied TECAN multi function measuring instrument infinite M200pro to examine the fluorescence intensity of GFP sample in different dilution buffers, and established the standard curve between fluorescence intensity and GFP relative content. Compared the results obtained from multi function measuring instrument, fluorescence-activated cell sorting （FACS） and Western-blotting. RESULTS： 50 mmol/L Tris-HCl （pH=10） was more suitable for examining GFP samples with low content than other dilution buffers. The regression coefficient R2 between fluorescent value and GFP content was above 0,99 by using multi function measuring instrument. The results obtained from multi function measuring instrument correlated significantly with those obtained from FACS and Western- blotting. CONCLUSION： The established method of GFP convenient, fast and could be used to screen large samples in a assay using multi function measuring instrument was short time.

关 键 词：绿色荧光蛋白 报告基因 高通量检测 多功能酶标仪 

分 类 号：Q291[生物学—细胞生物学]