Sample extraction techniques for enhanced proteomic analysis of plant tissues
Sample extraction techniques for enhanced proteomic analysis of plant tissues
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DOI：
10.1038/nprot.2006.102
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Rose, Jocelyn K. C.
中科院分区：
文献类型：
--
作者：
Isaacson, Tal;Damasceno, Cynthia M. B.;Rose, Jocelyn K. C.
近年来，蛋白质组学技术的重大改进使其在包括植物科学在内的所有生物学领域的应用增加。对于所有蛋白质组学方法，蛋白质提取和样品制备对于最佳结果至关重要;然而，从植物组织中提取蛋白质是一个巨大的挑战。植物组织通常含有相对少量的蛋白质和高浓度的蛋白酶和化合物，这些蛋白酶和化合物可能会限制组织分解并干扰随后的蛋白质分离和鉴定。有效的蛋白质提取方案还必须适应组织之间发生的次级代谢物和潜在污染化合物的巨大变化（例如，例如，在一个实施例中，叶、根、果实、种子和茎）和物种之间。在这里，我们提出了两个基本的蛋白质提取协议，已成功地用于不同的植物组织，包括柑橘组织。第一种方法是基于苯酚萃取耦合乙酸铵沉淀，第二种是基于三氯乙酸（TCA）沉淀。两种提取方案均可在2d内完成。
Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e. g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.