蛋白免疫印迹半定量分析在药理学研究中的影响因素

发布时间：2018-04-03 10:35

本文选题：蛋白免疫印迹　切入点：半定量　出处：《中国药学杂志》2015年09期

【摘要】：目的探讨蛋白免疫印迹半定量分析的影响因素,为在蛋白水平上研究药物的药理作用提供指导。方法提取丙型肝炎病毒(HCV)感染的Huh7.5细胞内总蛋白,蛋白定量后,以丙型肝炎病毒编码的核心蛋白为目的蛋白,以管家基因编码的蛋白Tubulin或Gapdh为内参对照,分析蛋白上样量、抗体浓度及孵育时间、发光液浓度等可变因素对蛋白免疫印迹半定量分析结果的影响。结果蛋白免疫印迹半定量结果中目的蛋白相对强度与蛋白总上样量之间只在一定上样量范围内有线性关系,超出线性范围会导致相对蛋白强度变化幅度减少,甚至无变化;上样体积、内参的选择不影响实验结果;在条带质量较好的前提下,一抗浓度及其孵育时间和发光液浓度也不改变实验结果;但是半定量有一定的缺陷,实验结果有一定的误差,应在15%以内。结论影响蛋白免疫印迹半定量结果的主要因素为目的蛋白的上样量;在能获得条带质量较好的前提下,改变上样体积和抗体孵育时间、适当稀释抗体和发光液浓度有利于实验进展而不影响实验结果。
[Abstract]:Objective to investigate the influencing factors of Western blot semi-quantitative analysis and to provide guidance for the study of pharmacological action of drugs at protein level.Methods the total protein of Huh7.5 cells infected with hepatitis C virus (HCV) was extracted. The core protein encoded by hepatitis C virus (HCV) was used as the target protein and the protein encoded by housekeeping gene (Tubulin or Gapdh) was used as internal control.The effects of antibody concentration, incubation time, luminescence concentration and other variable factors on the results of semi-quantitative analysis of Western blot.Results in the semi-quantitative results of Western blot, there was only a linear relationship between the relative strength of the target protein and the total amount of protein sample, and beyond the linear range, the change of relative protein intensity would decrease or even no change.The selection of sample volume and internal parameters did not affect the experimental results. Under the premise of good strip quality, the first antibody concentration, incubation time and luminescent solution concentration did not change the experimental results, but there were some defects in semi-quantitative analysis.There is a certain error in the experimental results, which should be within 15%.Conclusion the main factor influencing the semi-quantitative results of Western blot is the sample quantity of the target protein, and the volume of sample and the incubation time of antibody can be changed on the premise that the quality of the band can be obtained.The proper dilution of antibody and luminescent solution concentration is beneficial to the progress of the experiment without affecting the experimental results.
【作者单位】：中国医学科学院北京协和医学院医药生物技术研究所;中国医学科学院北京协和医学院药物研究所;
【基金】：国家自然科学基金委优秀青年科学基金资助项目(81322050) 教育部新世纪优秀人才项目(NCET-12-0072) 北京市科技新星项目(2010B072)
【分类号】：R96

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