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。

OBJECTIVE To investigate the impact factors of the semi-quantitative Western blot (WB), offering a guidance on research of pharmacological effects of drugs at the level of proteins. METHODS Total proteins were extracted from the Huh7.5 cells infected with hepatitis C virus and quantified with BCA kit, HCV Core protein was chose as target protein, and protein Tubulin or Gapdh, which was encoded by housekeeping gene, as the internal control. By controlling the experimental factors, such as loading sample amount, concentration and the incubation time of first antibodies, and dilutions of chemiluminescent fluid as well, we analyzed those factors how to impact the semi-quantitative results. RESULTS The semi-quantitative results showed that there is a linear relationship between relative intensity of target protein and the amount of total protein at must protein concentration range, beyond which, the rangeability of relative intensity of target protein reduced, even no changes. However, sample volume loaded, or protein selected as internal reference has little influence on the result of semi-quantitative WB. In the context of obtaining high-quality band, concentration and incubation time of antibodies or dilution of chemiluminescent fluid also has no influence on it. Yet, the semi-quantitative WB has certain defects and our results show that the permissible error of semi-quantitative result should be controlled within 15%. CONCLUSION The key impact factor on the result of semi-quantitative WB is the target protein amount of loading. On the premise of obtaining clear and high-quality bands, appropriately selecting loading volume of samples, concentration and incubation time of first antibodies, and dilutions of chemiluminescent fluid is conducive to accomplish experiments without affecting the final results of semi-quantitative WB.