通过对不同裂解液配方、等电聚焦程序和上样量等条件的对比试验,建立和优化了大黄鱼肝脏蛋白质组双向电泳的相关技术体系。试验结果表明,裂解液中添加Tris和TBP,聚焦时适当延长除盐时间,提高聚焦电压和功率,能显著提高双向电泳图谱的分辨率,而17 cm pH 5-8 IPG胶条的最佳上样量为2.0 mg。通过相关条件优化提高了大黄鱼肝脏蛋白双向电泳图谱的分辨率,为大黄鱼肝脏蛋白质组学的进一步研究奠定了基础。
The 2-DE related techniques was constructed and optimized in liver proteome of large yellow croaker Pseudosciaena crocea by comparative tests between different extraction methods,isoelectric focusing programs and sample volume.The results showed that the resolution of 2-DE profiles was significantly increased by adding Tris and TBP in lysis buffer,prolonging the time of desalting and increasing the voltage and power(Volt-hours)of isoelectric focusing properly.The optimal sample volume of 17 cm pH 5~ 8 IPG strips was found to be 2.0 mg.The 2-DE in liver proteome of the fish was successfully established with high quality map of separation under the optimization of the experimental conditions,which stands as a valuable resource for further liver proteomics research of Pseudosciaena crocea.