Western blot免疫印迹法检测磷酸化蛋白表达条件优化研究

Optimization Research on the Western Blot Experimental Conditions for Detecting Phosphorylated Protein

目的:探讨Western blot免疫印迹法不同转膜方法和不同抗原抗体比例对磷酸化蛋白表达的检测效果。方法:选择肌球蛋白轻链(myosin light chain,MLC)及其磷酸化蛋白作为研究对象,比较半干转印法、湿转法和1:3000、1:5000、1:10000等抗体稀释比例对磷酸化蛋白检测效果的影响。结果:半干转印法(恒压16V,30 min)观察到蛋白信号断续;而同样样品利用湿转法(恒压130 V,1h)检测发现信号连续且强度明显增高;对于磷酸化蛋白,半干转印法无法观察到磷酸化蛋白信号;而同样样品湿转法检测出现连续信号。统一利用湿转方法进行后续蛋白磷酸化检测,当抗体稀释比为1:3000时,结果出现非特异性条带;降低抗体稀释比为1:5000时无非特异性条带,且蛋白信号效果较好;抗体稀释比为1:10000时条带图像出现弥散且背景较高。结论:选择合适的转膜方式和抗原抗体比例有助于磷酸化蛋白表达检测。

Objective：Using different transfer methods and antigen-antibody dilution proportion to investigate the detection effects of Western blot on phosphorylated protein.Methods：Myosin light chain （MLC） and corresponding phosphorylated protein were chosen in the study.The conditions of different transfer methods （semi-dry transfer and wet transfer methods） and antigen-antibody dilution proportion （1∶ 3000,1∶ 5000,1∶10000） were compared.Results：The signals of β-actin and MLC from semi-dry transfer method （16 V,30 min） were discrete.However,signals from wet transfer method （130 V,1 h） were continuous and significantly stronger while using the same sample.There was no signal for the phosphorylated protein using semi-dry transfer method.The same sample showed a continuous signal using wet transfer method.Wet transfer method was applied for subsequent phosphorylated protein detection.Non-specific bands showed up when antigen-antibody dilution proportion was 1∶ 3000.Correct band showed up and non-specific bands disappeared when antigen-antibody dilution proportion was 1∶ 5000.The bands were diffused and background was high when antigen-antibody dilution proportion was 1∶10000.Conclusion：Choosing appropriate transfer methods and antigen-antibody dilution proportion can contribute to the detection effects ofphosphorylated protein using Western blot.