蝴蝶兰叶片蛋白质提取及双向电泳体系优化

摘要

A two-dimensional electrophoresis(2-DE)system for proteomic analysis ofPhalaenopsis leaves was established by optimizing the extraction methods, parameters including the pH gradient of IPG strips, sample loading, hydration ways and isoelectric focusing conditions. The optimized system included the following steps: extracting the total protein from Phalaenopsis leaves by phenol method, separating the proteins with 24 cm pH 3-10 NL IPG strips, loading protein samples of 1.35 mg by passive hydration followed by isoelectric focusing program B1, and staining the gels by Coomassie Brilliant Blue after SDS-PAGE (12%) electrophoresis. On the basis of optimized 2-DE system of Phalaenopsis leaves, reproducible profiles with high resolution were obtained.%通过对蛋白质提取、IPG胶条选择、上样量、水化方式、聚焦条件等方面的优化，建立蝴蝶兰叶片蛋白质的双向电泳体系。结果表明，采用酚抽提法提取蝴蝶兰叶片蛋白质的纯度较高，复溶较完全；双向电泳优化体系选用24 cm pH 3～10 NL的IPG胶条，被动水化，上样量为1.35 mg，B1程序进行等电聚焦，12%分离胶进行第二向电泳，考马斯亮蓝G-250染色。该方法获得分辨率较高、重复性较好的蝴蝶兰叶片双向电泳图谱，蛋白数点多达1163个，可以满足蝴蝶兰蛋白质组学研究和分析。