色谱

色谱

• 研究论文 • 上一篇    下一篇

一种基于二喹啉甲酸- Cu+显色反应的毛细管电泳检测蛋白质的新方法

孟庆威, 郭磊, 申睿, 谢剑炜*   

  1. 军事医学科学院毒物药物研究所, 北京 100850
  • 收稿日期:2010-03-05 修回日期:2010-05-05 出版日期:2010-07-28 发布日期:1980-03-25
  • 通讯作者: 谢剑炜

A novel capillary electrophoretic method for protein determination using bicinchoninic acid-Cu+colorimetric reaction

MENG Qingwei, GUO Lei, SHEN Rui, XIE Jianwei*   

  1. Beijing Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
  • Received:2010-03-05 Revised:2010-05-05 Online:2010-07-28 Published:1980-03-25
  • Contact: XIE Jianwei

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摘要： 采用毛细管电泳法和蛋白质显色反应-二喹啉甲酸(BCA)法,结合微波辅助反应,在60 mmol/L硼酸盐缓冲液(pH 9.5)中,实现了对蛋白质的快速毛细管电泳分析检测。同时以β-环糊精为包合添加剂,实现了BCA-Cu+复合物和游离BCA的分离,从而在波长200 nm处以测定特征生成的BCA-Cu+复合物来间接检测蛋白质,其峰强度比直接检测蛋白质自身吸收的峰强度提高了2个数量级。对于转铁蛋白、蓖麻毒素,其线性范围分别为2～200 mg/L和2～100 mg/L,检出限分别为0.33和0.37 mg/L。将该方法成功地应用于第一届蓖麻毒素国际实验室间比对测试的部分样品,含量测定结果满意。

关键词: β-环糊精, 蛋白质 , 二喹啉甲酸, 毛细管电泳, 显色反应, 亚铜离子

Abstract: A new rapid, sensitive method for protein determination using capillary electrophoresis and specific colorimetric reaction of bicinchoninic acid (BCA) was established, assisted by microwave incubation. With 60 mmol/L boric acid buffer (pH 9.5) and inclusion additive of β-cyclodextrin, the complex of BCA-Cu+ and free BCA molecules were efficiently separated. The peak intensity of BCA-Cu+was higher than those of native proteins about two orders of magnitude at a low wavelength of 200 nm. The linear ranges of this method were from 2 to 200 mg/L for transferrin, and 2 to 100 mg/L for ricin. The limits of detection for transferrin and ricin were 0.33 and 0.37 mg/L, respectively. This method was also successfully applied in the determination of some ricin samples in the First International Proficiency Test on the quantification of ricin.

Key words: bicinchoninic acid (BCA), colorimetric reaction, Cu+, protein , β-cyclodextrin, capillary electrophoresis (CE)