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适于双向电泳分析的苹果叶片蛋白质提取方法

曾广娟1,2, 李春敏2, 张新忠3, 滕云龙1, 董文轩1*   

  1. 1.College of Horticulture, Shenyang Agricultural University, Shenyang 110161, China; 2.Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Science, Changli 066600, China; 3.Institute for Horticultural Plants, China Agricultural University, Beijing 100193, China
  • 收稿日期:2009-02-11 修回日期:2009-04-10 出版日期:2009-07-30 发布日期:1982-06-25
  • 通讯作者: 董文轩

Efficient protein extraction method from apple leaves for apple proteomic analysis using two-dimensional electrophoresis analysis

ZENG Guangjuan1,2, LI Chunmin2, ZHANG Xinzhong3, TENG Yunlong1, DONG Wenxuan1*   

  1. 1.College of Horticulture, Shenyang Agricultural University, Shenyang 110161, China; 2.Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Science, Changli 066600, China; 3.Institute for Horticultural Plants, China Agricultural University, Beijing 100193, China
  • Received:2009-02-11 Revised:2009-04-10 Online:2009-07-30 Published:1982-06-25
  • Contact: DONG Wenxuan

PDF

755

被引次数

38

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7 cm、pH 3～10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18 cm、pH 3～10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14000～66000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。

关键词: 蛋白质提取方法, 蛋白质组学分析 , 苹果叶片, 双向电泳

Abstract: 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 616 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000~66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.

Key words: apple leaves, protein extraction method, proteomic analysis , two-dimensional electrophoresis (2-DE)