摘要
为了探索适用于双向电泳（2-DE）分析的苹果叶片蛋白质提取方法,比较了三氯乙酸（TCA）/丙酮沉淀法、二硫苏糖醇（DTT）/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7cm、pH3-10的线性固相pH梯度（immobilized pH gradient,IPG）胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）（12.5%的分离胶）作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18cm、pH3-10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000-66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。
In order to develop an efficient protein extraction method suitable for apple leaf proteomic analysis, four extraction methods for total protein in apple leaves were compared, including trichloroacetic acid （TCA）/acetone precipitation, dithiothreitol （DTT）/acetone method, tri（hydroxymethyl）aminomethane （Tris-HCl） method and the modified Tris-HCl method. During the two-dimensional electrophoresis （2-DE）, the first dimension electrophoresis was performed on a 7 cm strip with pH 3 - 10 linear immobilized pH gradient （IPG） and the second one was performed on 12.5% polyacrylamide gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The proteins were detected by silver staining. The results showed that 140, 215, 181 and 615 protein spots were detected on 2-DE gels, respectively. The modified Tris-HCl method was the most appropriate for apple leaf proteomic analysis because of the highest resolution and no apparent vertical or horizontal streaking on the 2-DE map. In order to testify the effect of the modified Tris-HCl method on the apple leaf protein extraction, 2-DE maps were established by using 18 cm strips with linear IPG in pH range of 3 - 10. After 2- DE separation and Coomassie Brilliant Blue R-250 （ CBB R-250） staining, about 455 spots were detected, and the relative molecular masses of most proteins were distributed in the range of 14 000 - 65 000 which were free of smearing or streaking. So it was once again proved that the modified Tris-HCl method can be used in apple leaf proteome analysis.
出处
《色谱》
CAS
CSCD
北大核心
2009年第4期484-488,共5页
Chinese Journal of Chromatography
基金
河北省自然科学基金项目(C2007000967)
关键词
双向电泳
蛋白质提取方法
苹果叶片
蛋白质组学分析
two-dimensional electrophoresis （ 2-DE ）
protein extraction method
apple leaves
proteomic analysis
作者简介
董文轩，教授，研究方向为果树种质资源与遗传育种．Tel：（024）88487143，E-mail：wxdong63@126．com．
