以金鱼胚胎为材料,以不同样品制备方式、裂解液成分、固相pH梯度胶条的选择、凝胶浓度为侧重点,建立并优化金鱼胚胎总蛋白质组最佳双向凝胶电泳技术条件。研究表明,采用匀浆器匀浆含硫脲和NP-40裂解液提取蛋白,应用pH值为3~10的非线性IPG胶条和浓度14%的SDS-PAGE凝胶进行双向电泳,经优化电泳电流、时间等参数后,获得了重复性和分辫率理想的金鱼胚胎总蛋白质双向电泳图谱,为金鱼胚胎蛋白质组分析奠定了基础,为揭示金鱼单倍体发育异常的机制提供了技术保障。
To develop and optimize a two-dimensional (2-DE) gel electrophoresis condition for gold{ish embryos proteome, total protein of goldfish embryos is extracted, and the optimal conditions for sample preparation and 2-DE are screened by comparing the following parameters: embryos broken methods, contents of lysis buffer, the concentration of gel, and linear or non-linear immobilized pH gradient gels. The results demonstrate that a satisfactory 2-DE map with good repetition and resolution is obtained and suitable conditions for 2-DE are established, with the conditions of the homogenate of goldfish embryos, adjusting IPG pH from 3 to 10 non-linear immobilized pH gradient gels and using 14 % SDS-PAGE gel as well as optimizing electrophoresis current, time and other parameters. The results paves a way for analysis of global protein profile in goldfish embryos, and lays a technological foundation for further study in the developmental mechanism of goldfish gynogenetic haploid embryos.