二硫苏糖醇和β-巯基乙醇在SDS-PAGE电泳过程中的扩散效应

Diffusing effect of DTT and 2-ME during SDS-PAGE electrophoresis

目的:二硫苏糖醇(DTT)和β-巯基乙醇(2-ME)是十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术中最常用的2种还原剂。文中旨在观察这2种还原剂在SDS-PAGE电泳过程中的扩散效应及建立规避扩散效应的方法。方法:将含有还原剂的蛋白样品和未还原蛋白样品间隔上样进行电泳,通过观察未还原样品的电泳条带来判断是否受到邻近泳道还原剂的扩散影响。结果:还原剂DTT或2-ME可扩散到邻近泳道,使未还原蛋白样品发生部分还原,扩散效应的强弱与还原剂的含量成正相关。结论:SDS-PAGE电泳过程中DTT和2-ME均存在扩散效应,在同一块凝胶上同时分离还原蛋白和未还原蛋白时,至少应该在2个泳道间设置一空白泳道,以规避扩散效应。

Objective： Dithiothreitol （DTT） and β-Mercaptoethanol （2-ME） are important reducing agents for SDS-PAGE. This study is to observe the diffusing effect of DTT and 2-ME during electrophoresis, and to find a way of avoiding this effect. Methods ： We placed protein samples containing reducing agents and non-reduced protein samples separately in the sample wells at intervals for SDS-PAGE electrophoresis, and determined whether or not the electrophoretic lanes of the non-reduced samples were interfered by the adjacent lanes. Results： DTT and 2-ME diffused to the neighboring lane, so that the nonreduced samples were reduced partially. The spreading effect was positively correlated with the content of the reducing agent. Conclusion ： DTT and 2-ME have a diffusing effect during SDS-PAGE electrophoresis. In separating the reduced and non-reduced proteins in the same gel at the same time, at least a blank lane should be set up in between them in order to avoid the diffusing effect.