Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis
Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis
复制标题
用于作物蛋白质组分析的三氯乙酸/丙酮沉淀与苯酚萃取相结合的通用样品制备方法
DOI：
10.1038/nprot.2014.022
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发表时间：
2014-02-01
期刊：
影响因子：
14.8
通讯作者：
Cresti, Mauro
中科院分区：
文献类型：
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作者：
Wu, Xiaolin;Xiong, Erhui;Cresti, Mauro
农作物含有大量的次生化合物，会干扰蛋白质提取和基于凝胶的蛋白质组分析。因此，一种能够以最小的优化方便地应用于各种作物材料的蛋白质提取方案是必不可少的。在这里，我们描述了一种通用的总蛋白提取方法，包括三氯乙酸(TCA)/丙酮沉淀，然后是十二烷基硫酸钠和苯酚提取。通过十二烷基硫酸钠(SDS)提取，TCA/丙酮沉淀的蛋白质可以完全溶解，然后用苯酚提取进一步纯化。该方案结合了TCA/丙酮沉淀法和苯酚提取法，前者能积极去除非蛋白质化合物，后者能选择性地溶解蛋白质，从而有效地从作物组织中提纯蛋白质。该方法还可以从各种顽固组织中制备高质量的蛋白质制剂，因此在作物蛋白质组分析中有广泛的应用。该协议设计为小规模运行，可以在5小时内完成。
Crop plants contain large amounts of secondary compounds that interfere with protein extraction and gel-based proteomic analysis. Thus, a protein extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and phenol extraction. Through SDS extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by phenol extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and phenol extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.