摘要
为建立适用于发菜（Nostoc flagelliforme）蛋白质组研究的双向电泳技术，对发菜蛋白质的提取、裂解、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化，结果显示：发菜蛋白质主要分布在pH4～7范围内，采用改良TCA法可提高提取液中蛋白质的含量和双向电泳图谱的分辨率，裂解液含60mmol／LDTT，24cm IPG胶条上样量1．5mg时不仅提高了蛋白质的溶解性，而且改善了双向电泳的分离效果，得到近800个蛋白点，且蛋白点清晰，图谱分辨率较好。采用优化后的双向电泳体系提高了发菜蛋白质双向电泳的分辨率和重复性，建立起一套适用于发菜蛋白质组分析的双向电泳方法。
In order to establish a two-dimensional gel electrophoresis （2D-E） protocol for proteomic study of Nostoc flagelliforme ,we optimized extracting method of total protein, lysis method, loading quantity and the key process of IEF and SDS-PAGE. The results showed that the protein spots were distributed mainly within the range of pH 4 -7. Improved TCA method was obviously improve the protein content and resolution patterns,lysis buffer contained 60 mmol/L DTT and loading quantity of sample protein were 1.5 mg/ （24 cm IPG trip） could obtain clear map and better separation effect.More than 800 protein spots with pH 4-7 were detected, by the way of staining with Colloidal coomassie blue R-250. The optimized protocol of N. flagelliforme markedly minimized the interference from non-protein substances.
出处
《西北植物学报》
CAS
CSCD
北大核心
2009年第8期1550-1556,共7页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(30760022)
关键词
发菜
双向电泳
蛋白质组学
Nostoc flagelliforrne
two-dimensional gel electrophoresis
proteomics
