甜瓜叶片中Rubisco的去除及双向电泳体系的优化

Depletion of Rubisco from Cucumis melon Leaf and Optimization of Two-Dimensional Electrophoresis for Proteome

为降低Rubisco的干扰,建立适合于甜瓜叶片蛋白质的双向电泳技术,本文比较了不同全蛋白提取方法和上样量对双向电泳的影响。结果表明,Mg/NP-40/PEG3350/TCA/丙酮提取法可去除样品中绝大多数的Rubisco,使低丰度蛋白得以检测,适合后续分析。以该法提取的蛋白,采用600、800、1000和l200μg四种上样量进行双向电泳,上样量为l000μg的样品用pH3～10的IPG胶条,在2-DE胶上分辨出质量好、数量多的蛋白质点(562个)。因此,Mg/NP-40/PEG3350/TCA/丙酮法是适合甜瓜叶片蛋白质提取的方法,l000μg是最适上样量。

This research compared the effects of different methods of protein extraction of Cucumis melon leaves and different loading amounts on 2-DE profile.The results indicated that Mg/NP-40/PEG3350/TCA/acetone precipitation was a suitable method for subsequent analysis attributed to reducing the interference of Rubisco.There were 562 proteins could be well resolved and detected in the gels when l 000 μg protein of leaves was loading with pH 3-10 IPG strips.All the results showed that Mg/NP-40/PEG3350 TCA/acetone precipitation was much more suitable for protein extraction and l 000 μg was the optimum loading amount of Cucumis melon leaves.