龙眼种子蛋白质组双向电泳技术的优化

Optimization of two-dimensional gel electrophoresis for proteome of longan seed

为建立适用于龙眼(Dimdcarpus longanaLour.)种子蛋白质组研究的双向电泳技术,对龙眼种子蛋白质的IEF、平衡时间及染色方法等关键步骤进行了优化。结果显示,龙眼种子蛋白质主要分布在pH4-7范围内,采用酚抽法,在聚焦达到50000 W.h的情况下能充分地聚焦,等电聚焦后经平衡缓冲液Ⅰ还原15 min,再经平衡缓冲液Ⅱ二次平衡20 min,考马斯亮蓝染色,24 cm IPG胶条上样量为1.2 mg时,在龙眼种子双向电泳图谱上检测到1000个多个蛋白点,蛋白点清晰,分辨率较好。本研究所建立的一套适用于龙眼种子蛋白质组分析的双向电泳方法分辨率和重复性较高,重复样品图谱间的相关系数在0.88-0.945之间。

In order to establish a two-dimensional gel electrophoresis（2-DE） protocol for proteomic study of longan（Dimocarpus longan Lour.） seed,we optimized the key process of IEF,equilibrium time and a different method of staining.The results showed that the protein spots were distributed mainly within the range of pH 4-7.Proteins were extracted using phenol extraction procedure.Before SDS-PAGE,the IPG strips were equilibrated for 15 min in equilibration buffer,followed by a further 20 min in equilibration buffer,gels were stained with Coomassie Brilliant Blue（CCB） R-250 and loading quantity of sample protein were 1.2 mg（24 cm IPG strip） could obtain clear map.At least 1000 protein spots were detected on the gels of longan seed.The optimized protocol of logan seed markedly impoved the resolution patterns and repeatability.The correlation coefficient between replicates varied from 0.88-0.945.