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双向电泳(2-DE)技术是研究蛋白组学的有效途径和重要手段,而在双向电泳第一向中使用不同pH梯度的IPG胶条,是对双向电泳技术研究的深入。利用改良的TCA/丙酮与Tris-饱和酚提取法,提取纯度更高的绿竹叶片全蛋白,并在双向电泳中使用不同pH梯度的IPG胶条,检测其对绿竹叶片双向电泳图谱的影响。结果表明,蛋白样品上样量为20μg时,在7 cm的pH3-10L、pH3-10NL、pH5-8L 3种胶条中,pH5-8L的胶条在双向电泳的凝胶图谱上显现的蛋白点数目最多,约有400个,而且蛋白点的分布较其他2个胶条的结果更为均匀,清晰度更高;pH3-10NL的胶条的图谱上显现的蛋白点要比同一跨度的pH3-10L的胶条更多;通过3种胶条的对比可以使绿竹叶片双向电泳的分辨率显著提高。对于在pH5-8L显现出来的4个蛋白点进行MALDI-TOF-MS质谱分析,成功鉴定出4个蛋白点,其中3个蛋白点在pH3-10L胶条的双向电泳中是没有显现出来的,这些蛋白对于植物生物功能的发挥具有意义。试验表明了对于一些低丰度蛋白及小分子量蛋白的检测和鉴定,可以利用pH梯度较小的固化胶条进行双向电泳试验。
Two-dimensional electrophoresis (2-DE) is an effective way and an important method to study proteomics. However, the use of IPG strips with different pH gradients in the first direction of two-dimensional electrophoresis is a further study on two-dimensional electrophoresis. The improved pure TCA / acetone and Tris-saturated phenol extraction method was used to extract the more pure green bamboo leaf protein, and the two-dimensional electrophoresis with different pH gradient IPG strips were used to detect the two-dimensional gel electrophoresis . The results showed that when the amount of protein sample was 20μg, the gel of pH5-8L exhibited the gel protein in two-dimensional gel electrophoresis in 7 cm pH3-10L, pH3-10NL, pH5-8L three kinds of strips The number of points is up to about 400, and the distribution of protein spots is more uniform and the definition is higher than that of the other two strips; the spots on the strips of pH 3-10NL show more spots than the same span of pH3 -10L strip more; through the comparison of three kinds of strips can make green bamboo leaf two-dimensional electrophoresis resolution was significantly improved. Four protein spots were successfully identified by MALDI-TOF-MS mass spectrometry for the four protein spots appearing at pH5-8L. Three protein spots were not observed in the two-dimensional electrophoresis of pH3-10L strips, These proteins have implications for the biological function of plants. Experiments have shown that for the detection and identification of some low abundance proteins and small molecular weight proteins, two-dimensional electrophoresis assays can be performed using immobilized strips of lower pH gradient.