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棉花学报  2011 

陆地棉花药蛋白质组分析中双向电泳技术体系的建立与优化

DOI: 1002-7807(2011)04-300-06, PP. 300-305

Keywords: 蛋白质组,双向电泳,陆地棉,花药

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Abstract:

建立了适用于陆地棉花药蛋白质组研究的双向电泳技术。以陆地棉花药为材料,采用液氮研磨的方法破碎组织,然后用三氯乙酸(TCA)/丙酮沉淀法和苯酚抽提结合甲醇/醋酸铵沉淀法提取蛋白质。采取载体两性电解质pH梯度等电聚焦/SDS-PAGE和固相pH梯度等电聚焦/SDS-PAGE双向凝胶电泳,对陆地棉花药总蛋白质进行了分离。通过对样品制备方法、等电聚焦方法、样品上样量等关键技术进行比较和优化,发现苯酚抽提结合甲醇/醋酸铵沉淀的方法较适合陆地棉花药蛋白质的提取,采用载体两性电解质pH梯度聚焦方法较pH梯度等电聚焦方法的效果好,用硝酸银染色时,上样量为500μg,得到的电泳图谱分辨率高、重复性好。

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