摘要
双向电泳技术是蛋白质组学研究的基本方法之一。果实由于富含糖、多酚、单宁和有机酸等物质,蛋白质的提取比其它植物组织更加困难。本文主要介绍不同果实蛋白质的提取、等电聚焦系统和凝胶染色技术,并建立了一套适用于桃、樱桃、苹果、芒果和冬枣等多种果实蛋白质组学的研究方法。结果表明,采用匀浆法和酚抽提法提取果实的蛋白质,裂解缓冲液2溶解蛋白质,并用固相pH梯度进行等电聚焦,可以获得背景清晰和分辨率高的凝胶图谱,具有较好的重复性,可用于果实蛋白质组学的研究。我们的研究结果显示,固相干胶条与IEF管胶相比,具有更加明显的优势。而不同的染色方法,对结果影响不大。
Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. In this study, several 2-D electrophoresis （2-DE） protocols were evaluated to establish a reference map for the fruit proteome. Three protein extraction procedures for 2-DE and different electrophoresis systems of isoelectric focusing （IEF）, as well as staining protocols, were compared with several fruit protein samples. We achieved satisfactory 2-DE patterns with well-resolved polypeptide spots throughout the gel by homogenization （Homo） and phenol （Phe） protein extraction protocols in combination with a lysis buffer of 7 mol·L^-1 urea, 2 mol·L^-1 thiourea, 4% （v/v） CHAPS, 1% （w/v） DTT, 0.2% （v/v） Nonidet P40, and 2% （v/v） carrier ampholvtes. As well. immobilized pH gradient（IPG）-IEF produced high-quality 2-DE gels that were free of smearing and streaking, and all staining protocols were suitable for protein visualization. These results suggest that the Homo and Phe protocols are highly effective with fruit tissues and that the combination IPG-IEF provides satisfactory 2-DE-based proteomic analysis of fruit tissues, which is very useful for deep investigation of biochemical changes in harvested fruit by a proteomic approach.
出处
《植物学报》
CAS
CSCD
北大核心
2009年第1期107-116,共10页
Chinese Bulletin of Botany
基金
国家自然科学基金(No.30671473
U0631004)
关键词
果实
凝胶染色
等电聚焦
裂解缓冲液
蛋白质提取
fruit, gel staining, isoelectric focusing, lysis buffer, protein extraction
