蛋白免疫印迹法检测小分子蛋白的实验条件优化研究

目的:蛋白免疫印迹法自发明以来被广泛应用于现代生物学研究中的蛋白质定性和半定量分析。为了提高蛋白免疫印迹法的检测效率,需针对不同蛋白的特性调节相关的实验条件参数。本文旨在探讨免疫印迹法不同参数对小分子蛋白检测效果的影响,从而优化并获得最佳实验条件。方法:比较不同转膜电压和时间、转移缓冲液甲醇含量、不同化学发光剂对小分子蛋白的检测效果。结果:选择20 V、10 min转膜电压和时间所获得的信号显著高于10 V、25 min转膜条件,选择含20%甲醇转移缓冲液所获得的信号显著高于无甲醇转移缓冲液,选择飞克级化学发光剂所获得的信号显著高于纳克级化学发光剂。结论:选用高电压、短时间组合,选择含20%甲醇转移缓冲液和飞克级化学发光剂信号均有助于小分子蛋白免疫印迹检测。

Optimization Research on the Western Blot Experimental Conditions for Detecting Small Molecule Proteins*

Objective:Since the invention of the Western blotting method, it has been extensively applied as a protein qualitative and semi-quantitative analysis method in modern biology research. In order to improve the efficiency of this method, adjustments of the related experimental parameters were needed according to the characteristics of different proteins. Our purpose is to investigate the effects of different parameters of the Western blot in detecting small molecule proteins, thus optimize and get the ideal experimental conditions.Methods:Comparing the effects of different transfer voltages and times, different methanol contents of transfer buffer and different chemiluminescence reagents of western blot technology.Results:The signal of small protein obtained at 20 V for 1 0 min was much higher than that transferred at 10 V for 25 min. The signal of the 20 % methanol in transfer buffer was significantly higher than that of the 0 % methanol in transfer buffer. The effect of chemiluminescence reagent at femtogram level was superior to that of the nanogram level.Conclusion:Choosing high voltage and short time, choosing transfer buffer containing 20 % methanol and chemiluminescence reagent at femtogram level could be helpful in the detection of small molecule proteins by using the western blot assay.