草鱼鳃组织蛋白双向电泳技术的建立及优化  

Establishment and optimization of 2-DE technique in gill protein of Ctenopharyngodon idella

作　　者：徐湛宁 李福贵[1] 陈杰[1] 蒋霞云[1] 邹曙明[1] XU Zhan-ning LI Fu-gui CHEN Jie JIANG Xia-yun ZOU Shu-ming(Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, Shanghai 201306, China)

机构地区：[1]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306

出　　处：《淡水渔业》2017年第5期14-20,46,共8页Freshwater Fisheries

基　　金："十二五"国家863计划主题项目(2011AA100403);上海高校知识服务平台(ZF1206)

摘　　要：为建立并优化草鱼鳃蛋白质组双向电泳的技术体系,实验将草鱼鳃经裂解处理后,通过固相p H梯度胶条进行一向等电聚焦和SDS聚丙烯酰胺凝胶垂直电泳进行蛋白分离,对不同的裂解液配方、蛋白纯化方法、IPG胶条的分离范围、上样量和染色方法等进行了探索和优化,并应用lmage Master 2D Platinum 7.0软件对电泳图谱进行了分析。结果表明:采用裂解液中添加DTT来取代Tris和TBP、选择分离范围为pH4-7的IPG胶条和150μg的主动水化上样,电泳结束后对凝胶进行硝酸银染色,获得了覆盖范围广、低背景、高分辨率、适合差异蛋白分析的双向电泳参考图谱。In order to establish and optimize the two -dimensional electrophoresis （2 - DE ） system for proteomic analysis on the gill of Grass carp （ Ctenopharyngodon idella） , protein samples of grass carp gill were separated by lysis. Immobilized pH gradient（IPG） strips was used to perform isoelectric focusing electrophoresis（the first dimension） , and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS -PAGE） served as the protein separation. By optimizing different lysate formulations and protein purification methods, different immobilized pH gradients （IPG） gel strips, as well as varied loading amount and staining methods were investigated. The proteins were successfully isolated from grass carp gill and were separated by 2 - DE. Image Master 2D Platinum 7.0 analysis software was applied to analyze the 2 - DE images. The results showed that the quality of 2 -DE profiles was significantly improved by replacing Tris and TBP with DTT in lysis buffer, loading 150 μg samples on the pH 4 -7 immobilized pH gradients （IPG） strips, and staining with silver nitrite. In conse-quence, Compared with other methods the 2D - gels profiles with wider pH molecular weight range, lower background noise, higher resolution and more applicable for variant analysis were obtained by using this method consequently.

关 键 词：草鱼(Ctenopharyngodon idella) 鳃蛋白质 双向电泳 蛋白质组学 

分 类 号：S917.4[农业科学—水产科学]