本文選題：蛋白免疫印跡　切入點：半定量　出處：《中國藥學雜志》2015年09期

【摘要】：目的探討蛋白免疫印跡半定量分析的影響因素,為在蛋白水平上研究藥物的藥理作用提供指導。方法提取丙型肝炎病毒(HCV)感染的Huh7.5細胞內(nèi)總蛋白,蛋白定量后,以丙型肝炎病毒編碼的核心蛋白為目的蛋白,以管家基因編碼的蛋白Tubulin或Gapdh為內(nèi)參對照,分析蛋白上樣量、抗體濃度及孵育時間、發(fā)光液濃度等可變因素對蛋白免疫印跡半定量分析結(jié)果的影響。結(jié)果蛋白免疫印跡半定量結(jié)果中目的蛋白相對強度與蛋白總上樣量之間只在一定上樣量范圍內(nèi)有線性關(guān)系,超出線性范圍會導致相對蛋白強度變化幅度減少,甚至無變化;上樣體積、內(nèi)參的選擇不影響實驗結(jié)果;在條帶質(zhì)量較好的前提下,一抗?jié)舛燃捌浞跤龝r間和發(fā)光液濃度也不改變實驗結(jié)果;但是半定量有一定的缺陷,實驗結(jié)果有一定的誤差,應在15%以內(nèi)。結(jié)論影響蛋白免疫印跡半定量結(jié)果的主要因素為目的蛋白的上樣量;在能獲得條帶質(zhì)量較好的前提下,改變上樣體積和抗體孵育時間、適當稀釋抗體和發(fā)光液濃度有利于實驗進展而不影響實驗結(jié)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.
【作者單位】： 中國醫(yī)學科學院北京協(xié)和醫(yī)學院醫(yī)藥生物技術(shù)研究所;中國醫(yī)學科學院北京協(xié)和醫(yī)學院藥物研究所;
【基金】：國家自然科學基金委優(yōu)秀青年科學基金資助項目(81322050) 教育部新世紀優(yōu)秀人才項目(NCET-12-0072) 北京市科技新星項目(2010B072)
【分類號】：R96

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