适用于小麦叶片总蛋白分析的双向电泳技术

Analysis of Total Protein in Wheat Leaf Using Two-dimensional Electrophoresis

双向电泳技术越来越成为植物蛋白质组研究中的关键技术,样品制备、固相预制胶条水化、等电聚焦及SDS-PAGE都是影响双向电泳结果的重要步骤,本研究以晋麦47号小麦叶片为研究材料,在样品除盐、预制胶条水化方式、第二向SDS-PAGE电压选择方面进行了探索与优化,通过比较双向电泳图谱,建立适用于小麦叶片总蛋白质分析的双向电泳技术体系.结果表明:样品经多步除盐并加长除盐时间后进行双向电泳,蛋白能较好地被分离,2-DE图谱蛋白点较多,分辨率较高;IPG胶条先后经被动水化与主动水化比仅主动水化的2-DE图谱蛋白点损失少;第二向SDS-PAGE中电压选择120 V较200 V的2-DE图谱蛋白点拖尾少,纹理现象少.

Two-dimensional electrophoresis（2-DE） has increasingly become one of the key technology in the study of plant protein. Sample preparation, 1PG strip rehydration, isoelectrie focusing electrophoresis and SDS- PAGE are the important steps in 2-DE. The conditions affecting 2-DE, have been optimized, such as the precess of sample desalting, the way of 1PG strip rehydration and the vohge of SDS-PAGE. And then the 2-DE system are establised for analysing wheat leaf total protein in Jinmai 47. The results show that the proteins could be well separa- ted after multi-step desalting and extension of the desahing time. Many protein points and high resolution occur in 2-DE; the loss of protein points through passive hydration and active hydration is less than that of only active hydration for IPG strip; the tailing and texture phenomenons using 120 V voltage are less than that of 200 V voltage in 2-DE diagram.