蛋白免疫印迹法同时检测大、小分子蛋白的实验条件改进

目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。

Improvement on the Western Blot Experimental Conditions for Simultaneous Analysis of Proteins with High and Low Molecular Mass

Objective:Western blot is widely used for identifying and semi-quantifying proteins in modern biomedicine research. However, there are some limitations in conventional method using single-concentrated gel such as it can''t detect proteins with high and low mass simultaneously. Therefore, it is important to explore a modified method with a wider range of separation. Here, we propose a combination gel made up of two different concentrations of separating gels for improvement in simultaneous analysis of proteins in wider range of molecular weight.Methods:Comparing the separation range between combination gel of two different concentrations and single-concentrated gel and detection of both proteins with high and low mass by western blot using combination gel.Results:Both 12% /7.5% combination gel and 15%/7.5% combination gel resolved wider ranges of proteins than that of single-concentrated gel. What is more, using 12%/7.5% combination gel, it simultaneously detected target proteins within the mass rang of 15-300 kDa by Western blot.Conclusion:Combination gels improve simultaneous analysis of proteins that differ greatly in size by Western blot and may be widely applied in the future.