银杏小孢子叶球蛋白提取和双向电泳体系优化(Ⅱ)  

Protein Extraction from Ginkgo biloba Starninate Strobilus and Optimization of Two-Dimensional Electrophoresis Protocol

作　　者：杨妮娜[1] 崔彬彬[2] 孙宇涵[1] 李允菲[1] 王亚茹[1] 陈欣[1] 李云[1] 邓高松[3] 苗少波[3] 

机构地区：[1]北京林业大学,北京100083 [2]保定学院 [3]北京林业大学实验林场

出　　处：《东北林业大学学报》2011年第8期55-57,共3页Journal of Northeast Forestry University

基　　金：国家自然科学基金(30771747);林业行业专项(201004008)

摘　　要：以银杏小孢子叶球为材料,比较了不同的提取的方法、蛋白质裂解液组成、pH值范围以及不同质量浓度SDS-PAGE凝胶对双向电泳的影响。结果表明:TCA/丙酮/酚法比TCA/丙酮法更适合于银杏小孢子叶球蛋白的提取;含有硫脲的蛋白质裂解液Ⅱ,可显著提高蛋白质的溶解性,在2-DE图谱上可分辨出748个蛋白质点,比用蛋白质裂解液Ⅰ提取的蛋白点数多620个。采用pH3-10的胶条分离时,蛋白点集中在pH=4～8,使用pH=4～7的胶条,图谱胶点重叠少,也较清晰,更利于低丰度蛋白的检出。用TCA/丙酮/酚提取蛋白,并以蛋白质裂解液Ⅱ溶解蛋白,采用pH=4～7线性17 cm的IPG胶条1,2%SDS-PAGE凝胶进行双向凝胶电泳,银杏小孢子叶球蛋白质能得到较好的分离,图谱清晰。The effects of different protein extraction methods, lysis buffers, pH ranges of IPG strip and different gel concentrations on two-dimensional gel clectrophoresis were studied by using male flower buds of Ginkgo biloba as experimental materials. Result shows that, compared TCA-acetonc-phenol protocol with TCA-acetone protocol, the former is more suitable. The protein solubility of the samples dissolved in lysis buffer Ⅱ containing thiourea was greatly enhanced, and about 748 protein spots were detected on 2-DE gels, which was 620 more than that in lysis buffer Ⅰ. More spots were assembled from pH 4 to pH 8. Replaced pH 3-10 strips with pH 4-7 strips, the spots were shown in a clearer way. When the proteins were extracted by the TCA-acetone-phenol method, dissolved with the lysis buffer Ⅱ, with 17 cm, pH 4-7 IPG strip, and 12% concentrations of the SDS-PAGE gel, they were better separated.

关 键 词：银杏 TCA/丙酮/酚 双向电泳技术 

分 类 号：S792.95[农业科学—林木遗传育种]