基础医学与临床 ›› 2017, Vol. 37 ›› Issue (10): 1378-1383.

• 研究论文 • 上一篇    下一篇

微流控蛋白印迹杂交与传统Western blot对目的蛋白的检测效果比较

辛晓磊1,慕轩2,王欣3,刘力4   

  1. 1. 中国医学科学院基础医学研究所
    2. 中国医学科学院 北京协和医学院 基础学院
    3. 中国医学科学院 基础医学研究所 北京协和医学院 基础学院
    4. 中国医学科学院基础医学研究所 北京协和医学院基础学院
  • 收稿日期:2017-07-07 修回日期:2017-07-11 出版日期:2017-10-05 发布日期:2017-09-25
  • 通讯作者: 刘力 E-mail:liliu@pumc.edu.cn
  • 基金资助:
    国家自然科学基金;国家自然科学基金

Comparison of the effects of microfluidic paper-based immunoassay and traditional Western blot on the detection of target proteins

  • Received:2017-07-07 Revised:2017-07-11 Online:2017-10-05 Published:2017-09-25

摘要: 目的 借助微流控蛋白印迹实验实现对目的蛋白省时、省力和省试剂的定性以及相对定量分析。方法 选取O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)蛋白为目的蛋白,选用高表达MGMT蛋白的MCF7细胞为细胞模型,用传统蛋白印迹杂交和纸芯片蛋白印迹杂交实验法对经过药物—MGMT阻断剂(lomeguatrib)处理后MCF7细胞的MGMT蛋白表达量做定性以及相对定量分析。结果 纸芯片蛋白印迹杂交可以成功进行MGMT的定性、相对定量分析,而且上样检测量可低至10~25pg。这相比于传统Western blot的上样检测蛋白检测限1~5ng,提高了3个数量级。整个操作流程只需要1h,所用试剂量少,实验成本更低。而且,纸芯片的多通道构造可以实现如同反向蛋白杂交分析(RPPA)一样的,对组织蛋白表达量进行高通量的系统检测。结论 微流控蛋白印迹杂交相较于传统Western blot 可以检测到更微量级的蛋白,节省试剂和样品,省时和省力,并可进行高通量的定性以及相对定量分析。

关键词: 关键词:微流控纸芯片, Western blot, MGMT, 定性检测, 相对定量检测

Abstract: Objective To achieve the goal of the qualitative and relatively quantitative analysis of the target protein in a time-saving, labor-saving and reagents-saving way by the microfluidic paper-based immunoassay. Methods In this paper, we chose MGMT as the target protein and compared the qualitative and semi-quantitative results of the MGMT expression in the MCF7 cells which was treated with MGMT inhibitor, Lomeguatrib, in both the traditional Western Blotting and the paper chip immunoassay. Results Microfluidic paper-based immunoassay can make the qualitative and relative quantitative detection on protein expression. Compared to the sensitivity of 1-5ng of the traditional Western Blot, the microfluidic paper-based immunoassay could detect as low as 10-25 pg of the protein. The sensitivity could be improved by 3 orders of magnitude. The entire operational duration took only 1 hour with less costed reagents being consumed. It could make the high-throughput protein detection as sensitive as the reverse phase protein assays (RPPA) does. Conclusions The paper chip immunoassay could be performed qualitatively and semi-quantitatively to detect protein expression, and is more effective than that of traditional Western blot.

Key words: Key words:microfluidic paper-based immunoassay, Western Blot, MGMT, qualitative detection, relative quantitative detection

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