小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。

Optimization of Two-Dimensional Gel Electrophoresis for Proteins from Wheat Young Panicle

In this study,using common wheat BNS as experimental material,wheat protein sample of young panicle prepared by the methods of TCA-acetone extraction and phenolic extraction,respectively.Two-dimensional electrophoresis for the protein was carried out and problem occurred in the experimental procedure was discussed.The results showed that the protein yield extracted by TCA-acetone extraction method was much higher than that by phenolic extraction method.The SDS-PAGE electrophoresis demonstrated that the protein isolated by TCA/acetone extraction method exhibited clearer bands and higher resolution than that by the phenol extraction method with a fuzzy,low resolution.The prepared protein being desalination could improve the resolution and reduce the horizontal and vertical veins,and then obtained clear background round protein spots.By using ImageMasterTM 2D Platinum5.0 software to analyze the gel map,the results showed that protein purification could reduce the noise background and the number of protein spots after being purification could be elevated up to 583 from the 216 spots without purification.Clearly,using the TCA/acetone method can obtain the protein with high concentrations and high-quality,while further purification and desalination can further produce background clearly and reproducible polyacrylamide gel electrophoresis map.In the process of the two-dimensional electrophoresis experiments,we found some problems of the abnormal defective gels,such as diffusion of protein spots,protein aggregation to form a string spots,no spot or less spots,vertical and horizontal stripes and map distortions.Accordingly we had these serious problems analyzed and proposed the solutions.