猪肌肉组织双向电泳分离条件的建立及常见问题分析  

Establishment and Problem Analysis of Two-dimensional Electrophoresis Separation on Porcine Skeletal Muscle

作　　者：徐永杰[1,2] 熊远著[2] 

机构地区：[1]信阳师范学院生命科学学院遗传学研究所,信阳464000 [2]华中农业大学农业部猪遗传育种重点开放实验室,武汉430070

出　　处：《农业生物技术学报》2012年第1期105-112,共8页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2006CB102102);转基因生物新品种培育重大专项(No.2008ZX08006-002);信阳师范学院青年骨干教师资助计划(2011)

摘　　要：为建立和优化猪肌肉组织蛋白质双向电泳技术,从提取方法、加样量、IPG胶条的选择、SDS-PAGE胶浓度、染色方法等多个方面对双向电泳分离效果进行比较研究。结果显示,液氮研磨+超声破碎法提取猪肌肉组织蛋白效果优于液氮研磨法,前者细胞破碎彻底蛋白质溶解性好且核酸污染少,图谱质量较好;18cmpH4～7的IPG胶条分离效果比pH3～10非线性IPG胶条好;对于银染,18cmpH4～7的IPG胶条150μg上样量比较适宜;同一肌肉样品的三块胶的重复性可达70%以上;同时,对双向电泳过程中的典型问题作了详尽分析。研究结果表明,通过优化的双向电泳条件获得了较高分辨率和较好重复性的猪肌肉组织双向电泳图谱,可用于后续蛋白质组学分析。To establish two-dimensional gel electrophoresis methods（2-DE） for porcine skeletal muscle proteome,the experimental conditions of 2-DE was optimized,and various extraction methods,different loading quantities and pH IPG strips,different adhesive concentration and gel staining were studied.The results showed that the process of grinding in liquid nitrogen and following by ultrasonication was more appropriate than that of grinding in liquid nitrogen,which could obtained better 2-DE map for low nucleic acid contamination and more protein spots.Compared with different pH IPG strips,18 cm pH 4~7 IPG strips were better than pH 3~10 NL.For silver staining of 18 cm pH 4~7 IPG strip,the suitable loading quantity was 150 μg.Furthermore,the reproducibility of three gels in same sample was more 70%.Meanwhile,the common problems occurring in 2-DE experiments were analyzed.The results indicated that the 2-DE maps of porcine skeletal muscle were obtained with high resolution and reproducibility,and can be used for the further proteome analysis.

关 键 词：猪 肌肉 双向电泳 蛋白质组学 分析 

分 类 号：TS251.1[轻工技术与工程—农产品加工及贮藏工程]