运用统计学优化A型肉毒毒素的SDS-PAGE染脱色方法

Optimization of methods for staining and decoloration in SDS-PAGE of Botulinum toxin type A by statistic theory

目的运用统计学优化A型肉毒毒素的SDS-PAGE染色和脱色方法,以便生产检定中实现数字化、标准化操作。方法以Photoshop软件对电泳凝胶扫描图采样的背景灰度值、强带灰度值和弱带灰度值作为评价指标,以不同的脱色液为实验因素,按配对设计安排实验,对实验结果进行脱色方法的统计学分析,根据配对设计分析结果,按裂区设计安排第二步实验,对实验结果进行染色和脱色方法的以统计学为主的综合分析评价。结果综合分析显示,电泳凝胶直接由考马斯亮蓝R250-磷酸-乙醇染色液加热至60℃染色2 h,0.5 mol/L KCl结合冰醋酸-甲醇脱色可获得良好效果。结论得到一种具有一定创新性的A型肉毒毒素的SDS-PAGE染脱色方法,该方法具有背景脱色浅淡均匀,条带清晰,染色和脱色液不挥发因而重复性好的优点,能够满足A型肉毒毒素生产检定的需要。

Objective To optimize the methods for staining and decoloration in SDS-PAGE by statistic theory so as to digitize and standardize the operation in production process of Botulinum toxin type A. Methods Serving various destainers as experimental factors, the method for decoloration was evaluated with grey levels of background, strong bands and weak bands on the Photoshop scanning image by paired design, based on which the methods for staining and decoloration were analyzed by split plot design. Results Comprehensive analysis showed that the method was optimized as staining the electrophoretic gel with Coomassie brilliant blue R250-phosphoric acid-ethanol stainer by heating to 60 ℃for 2 h, followed by decoloration with 0. 5 mol / L potassium chloride combined with glacial acetic acid-methanol distaining solution. Conclusion An innovative method for staining and decoloration in SDS-PAGE of Botulinum toxin type A was developed, by which the background of gel was uniform, the bands were clear, and the reproducibility was improved because of nonvolatile solution. The method met the requirements for production and quality control of Botulinum toxin type A.