Tricine-SDS-PAGE分离鼻咽癌血清多肽方法初探

Separation of Peptides in Sera of Nasopharyngeal Carcinoma Patients with Tricine-SDS-PAGE System

目的建立鼻咽癌（nasopharyngeal carcinoma,NPC）血清多肽稳定且灵敏的Tricine-SDS-PAGE电泳体系。方法通过Tricine-SDS-PAGE电泳分离鼻咽癌血清多肽,比较乙腈处理与非处理血清,以及电泳时间长短等条件对分离效果的影响。结果鼻咽癌原始血清样本量200μL、经1.2倍体积乙腈沉淀,冷冻干燥后加入40μL的生理盐水溶解,取12~18μL上样,6.7%浓缩胶、夹层胶60 V,之后60 V的电压至电泳结束,电泳时间4 h 20 min,分离10 kDa以下的鼻咽癌血清多肽效果最佳。结论建立鼻咽癌血清多肽的Tricine-SDS-PAGE稳定电泳分离方法,电泳效果与样本的处理方法和电泳条件有关。

Objective To establish an effective Tricine-SDS-PAGE system for separating peptides from sera of patients with nasopharyngeal carcinoma.Methods Human serum albumin was removed by acetonitrile,and then peptides in sera of nasopharyngeal carcinoma patients were separated using Tricine-SDS-PAGE system.Comparisons of different electrophoresis conditions（including different electrophoresis time） were made for choosing the best electrophoretic system.Results The optimized Tricine-SDS-PAGE was performed as follows：serum samples（200 μL） from patients with nasopharyngeal carcinoma were depleted by acetonitrile（1.2 times the volume of samples） precipitation.After freeze-drying,the samples were mixed with 40 μL of saline solution.The supernatant（12~18 μL） was analyzed by Tricine-SDS-PAGE with 6.7% stacking gel at 60 V（initial voltage 60 V） for 4 hours and 20 minutes.Optimizedly,the peptides from sera of nasopharyngeal carcinoma patients（smaller than 10 kDa） could be stably separated using the Tricine-SDS-PAGE.Conclusion The stable Tricine-SDS-PAGE system for separating peptides from sera of nasopharyngeal carcinoma patients is correlated with methods for dealing with samples and conditions of electrophoresis.