Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling
Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling
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与蛋白质组学分析兼容的棉苗蛋白质提取方法
DOI：
10.2135/cropsci2008.06.0367
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发表时间：
2009-03-01
期刊：
影响因子：
2.3
通讯作者：
Yang, Xingyong
中科院分区：
文献类型：
--
作者：
Xie, ChengJian;Wang, De;Yang, Xingyong
高效的蛋白质提取方法是蛋白质组学研究的关键步骤。本研究的目的是比较四个先前公布的蛋白质提取方案，以确定一个合适的蛋白质提取方法，从棉花（Gossypium hirsutum L.）幼苗，一种柠檬状的植物组织。结果表明，三氯乙酸/丙酮沉淀结合苯酚提取法（方法D）的样品纯度最高，蛋白质斑点数最多（321个），其中碱性蛋白斑点最多。采用丙酮沉淀-苯酚提取法（方法B），在2-DE图谱上得到216个斑点。三氯乙酸/丙酮沉淀法（方法A）不适用于棉花幼苗，因为在2-DE图像中只能看到小分子量蛋白。苯酚提取法（方法C）在2-DE凝胶中显示240个斑点，但提供了更高的凝胶背景。随后，我们选择并优化了方法D从棉花幼苗中提取蛋白质。优化后，在pH 3-10非线性梯度条带双向凝胶电泳和考马斯亮蓝G-250染色的10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上检测到900多个斑点。我们的研究结果表明，优化后的方法D有望在棉花幼苗蛋白质组学研究中有很好的应用。
An efficient protein extraction method to ensure successful separation by two-dimensional electrophoresis (2-DE) is a critical step for any proteomics study. The objective of this study was to compare four previously published protein extraction protocols to identify a suitable protein extraction method to extract total proteins from cotton (Gossypium hirsutum L.) seedlings, a recalcitrant plant tissue. The results suggested that trichloroacetic acid/acetone precipitation combined with phenol extraction method (Method D) produced the purest sample and the most proteins spots (321), particularly basic proteins in the 2-DE image. The extraction method combining acetone precipitation and phenol (Method B) gave 216 spots in the 2-DE image. The trichloroacetic acid/acetone precipitation method (Method A) is not suitable for the cotton seedlings because only small molecular weight proteins were visualized in the 2-DE image. The phenol extraction method (Method C) showed 240 spots in the 2-DE gel but gave higher gel background. We subsequently selected and optimized Method D to extract protein from cotton seedlings. After optimization, more than 900 spots were detected on the 2-DE gel with pH 3-10 nonlinear gradient strip and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel using Coomassie brilliant blue G-250 staining. Our results suggest that the optimized Method D is expected to have excellent applications in proteomic studies of cotton seedlings.