大鼠肝组织中P-糖蛋白的免疫印迹检测方法

Detection of P-glycoprotein in Rat liver with Western Blotting

目的探讨Western Blot检测P-糖蛋白表达的实验优化条件。方法随机选择6只雄性SD大鼠,给予荭草花提取物0.4g/(kg·d)灌胃,连续灌胃6d。采用钙盐沉淀法提取6只大鼠肝微粒体,利用BCA法进行蛋白定量后,制备蛋白上样样品。比较不同样品处理方法、转膜方法的检测效果。结果与高温变性相比,样品经室温变性上样可检测到目的条带。湿法电转法较半干法电转法转膜效果好,且湿转条件选择120V,2h较为好。结论样品室温变性处理、120V,2h的湿转转膜条件,是检测P-糖蛋白较优的Western Blot方法。并且在经荭草花提取物给药后的6只SD大鼠肝组织内P-糖蛋白的表达呈现一定的差异。

Objective To determine the optimum experiment condition of detecting P-glycoprotein with western blotting（WB）.Methods 6 male SD rats were selected randomly and fed the Polygonumorientale Flower Extract with the dosage of 0.4 g/（kg·d）.The livers of 6 SD male rats were taken out and homogenated.Themicrosome was isolated by calcium ion precipitating and detected by BCA method.It was compared the effects of different protein treatments and different electrotransfer conditions.Results Only solubilizing at room temperature did obtained the destined protein compared with solubilizing at high temperature.It was more efficiency to use wet electroblotting which at constant voltage density of 120 Vfor 2 h.Conclusion Choosing samples denatured at room temperature,choosing wet electroblotting could be more repetitive and sensitive in the detection of P-glycoprotein by using the western blot（WB）assay.The expression of P-glycoprotein in liver of these six rats given Polygonumorientale FlowerExtract shows certain difference.