大葱花蕾总蛋白双向电泳体系的建立

Establishment of Two-dimensional Electrophoresis System for Buds of Welsh Onion

为建立适合于大葱花蕾总蛋白的双向电泳体系,本研究以大葱花蕾为材料,对大葱花蕾总蛋白提取方法、IPG胶条梯度范围、上样量、等电聚焦等双向电泳的关键步骤进行优化。结果表明最适宜大葱花蕾总蛋白的双向电泳条件为:TCA-丙酮沉淀法提取大葱花蕾总蛋白,选择24 cm p H 4~7的IPG胶条,上样量800μg,按聚焦程序Ⅰ聚焦后进行双向电泳并采用考马斯亮蓝染色。采用该体系可以获得分辨率高、重复性好的双向电泳图谱。

To establish a two-dime nsional electrophoresis system we used Welsh onion buds as materials, to optimize the method of the total protein extraction, the p H gradient of IPG strip, the sample loading and the isoelectric focusing conditions. Our results showed that: the best way to extract the total protein from Welsh onion flower was TCA/acetone precipitation; the ideal state of protein isolated included the 24 cm IPG strips by p H 4~7,800 μg loading samples, isoelectric focusing programⅠand staining with colloidal Coomassie Brilliant Blue after SDS-PAGE electrophoresis. The optimized 2-DE system of Welsh onion buds could obtain The high resolution and repetitive profiles.