几种双向凝胶电泳蛋白质检测方法的比较

几种双向凝胶电泳蛋白质检测方法的比较作者：秦慧，刘霆，柳斌，宋鑫，黄欣，杨金亮，赵霞，魏于全

【摘要】本研究对蛋白质染色法进行比较和分析，以求获得满足蛋白质组研究所需的适宜的蛋白质检测法。实验采用2-D电泳分离急性早幼粒白血病细胞株NB4的全蛋白及1-D电泳分离蛋白质分子量标准物，从蛋白质检测的灵敏性、质谱兼容性、操作的简便性等角度，比较了传统考马斯亮蓝染色法、胶体考马斯亮蓝染色法、改良考马斯亮蓝染色法和银染法4种蛋白质着色法对凝胶分离蛋白质的检测影响，探讨了影响蛋白质染色与鉴定的影响因素。结果显示，银染检测灵敏度最高，但质谱鉴定相容性差，鉴定率近10%；改良考马斯亮蓝染色法检测灵敏度也高，可达4 ng/spot，质谱鉴定率可接近55%。结论：改良考马斯亮蓝染色法的蛋白质检测灵敏度高，与质谱兼容好，能满足蛋白质组研究需要。

【关键词】蛋白质组

Abstract The aim of this study was to compare and analyze protein staining in order to select the optimal staining method for proteomic research. Proteins from acute promyelocytic

leukemia cell line NB4 and protein molecular weight marker were separated respectively by 2-D or 1-D electrophoresis and detected respectively by the typical Coomassie brilliant blue，the colloidal Coomassie brilliant blue，the modified Coomassie brilliant blue and the silver staining protocols. The protein detection sensitivity，compatibility with mass spectrometry (MS) and facility of the four staining protocols were compared. The results indicated that the silver staining exhibited the highest sensitivity and MS showed the lowest compatibility 10% of protein identification rate. The detection sensitivity of the modified Coomassie brilliant blue staining was superior to that of other two Coomassie brilliant blue stainings，close to but lower than the silver staining，however the compatibility with MS was better （protein identification rate about 55%）. It is concluded that the protein detection sensitivity of the modified Coomassie brilliant blue staining is high，and its compatibility with MS is better，this modified Coomassie brilliant blue staining is an optimal staining method for proteomic research.

Key words proteomics；two-dimensional gel electrophoresis；protein detection；Coomassie brilliant blue staining；silver staining

蛋白质组研究要求有高分辨率的蛋白质分离及准确、灵敏的质谱鉴定技术。凝胶电泳中蛋白质的着色不仅影响蛋白质分离的分辨率，同时也影响后续的质谱鉴定。选择适当的蛋白质染色法将有助于提高蛋白质组学研究结果的质量。蛋白质的染色常用的有4类：有机试剂染色、银染、荧光染色及同位素显色。其中有机试剂染色以考马斯亮蓝染色法（Coomasie brilliant blue，CBB）为代表，在蛋白质分析中常用，但对低丰度蛋白质的显现较差；银染灵敏度虽高，却常与质谱不兼容；荧光染色以SYPRO试剂为主，蛋白质检测灵敏度高，能兼容质谱，但由于需要配备特殊的检测仪器及试剂的昂贵，未被作为常规方法使用；而同位素显色则存在安全性和操作局限性等问题［1-4］。因此，筛选简便、节约、检测灵敏度高、质谱兼容的蛋白质着色法是蛋白质组研究所需。由于考马斯亮蓝染色法的广泛运用，近年来就考马斯亮蓝染色法在提高其灵敏性方面研究者们作了许多改进，方法众多，评价不一［5-7］。我们在作双向凝胶电泳时，将常用的几种考马斯亮蓝染色

法及银染进行了比较，并就其染色影响因素作出分析。

材料和方法

试剂