红外荧光标记蛋白检测方法的优化

Optimization of Western blot methods detected with infrared fluorescence

目的应用Odyssey双色红外荧光扫描成像系统定量检测蛋白质,通过对免疫印迹中转印膜和封闭液的筛选及抗体双标记的应用,优化红外荧光标记的蛋白检测方法。方法①差速离心法提取大鼠心肌组织膜蛋白,在转膜和封闭过程中比较了4组方案,通过对不同方案蛋白转印效率和背景噪音强度的差异比较,筛选最佳转印膜和封闭液;②运用抗体双标记的方法来研究不同种或不同形式蛋白在同一泳道的检测情况。结果①PVDF膜和脱脂奶粉封闭液结合应用,蛋白显现更清晰;②双重标记间隙连接蛋白43,同时检测到该蛋白两种形式。结论VDF膜、脱脂奶粉封闭液及抗体双标记的使用可以取得更好的红外荧光标记的蛋白检测结果。

Objective To optimize protein detection method by analysis of infrared fluorescence labling of Western blot to screen a transferring membrane and blocking solution for best result of Western blot. Methods ①Membrane proteins were extracted from rat myocardium. Four strategies with different transferring membrane and blocking solution were. compared during Western blot. ② Different types of connexin 43 in the same membrane were studied by applying double-labling antibody. Results ①Combination of PVDF and non-fat milk was the optimized material to get better Western blot result. ②Through double labling technique, two kinds of. connexin 43 were successfully detected. Conclusion Combination of to PVDF and non-fat milk can get better Western blot result.