利用TrueBlot抗体改善内源性蛋白的免疫沉淀/免疫印迹分析

摘要

目的：改善内源性蛋白的免疫沉淀/免疫印迹分析中免疫沉淀抗体对免疫印迹检测带来的干扰。方法：收集培养的Hela细胞或FLAG标记的S5b基因表达载体转染的Hela细胞，制备全细胞裂解液，分别加入特异性抗体沉淀S5b并洗脱，收集免疫复合物，通过SDS-PAGE分离，采用HRP标记的TrueBlot抗体或常规二抗，对S5b及其相互作用蛋白(Rpt1和Rpt2)进行免疫印迹检测。结果：得到了清晰的S5b，Rpt1和Rpt2条带。结论：TrueBlot抗体能明显消除免疫沉淀抗体的干扰，是内源性蛋白免疫沉淀后进行免疫印迹分析的理想工具。%Objective:To improve the immunoblotting of immunoprecipitated proteins and decrease the interference of immunoprecipitation antibody in the interaction of endogenous proteins. Methods:Transient transfect cells with fusion protein expression vector containing the targeted S5b gene and the FLAG tag, the transfected cells or untransfected cells were harvested to study the exogenous or endogenous protein interaction. The total cell lysate was immunoprecipitated by specific antibody. Then the eluted immunocomplex was separated by SDS-PAGE, and the TrueBlot antibody or conventional antibody was used as the secondary antibody for immunoblotting detection of S5b and its partner (Rpt1 and Rpt2). Results:Clear immunoblotting bands for S5b, Rpt1 and Rpt2 were obtained. Conclusion:TrueBlot antibody prefers the immunoblot antibody to immunoprecipitation antibody, and decreases the interruption of immunoprecipitation antibody to display clear protein band.