衫木叶片蛋白质组的双向电泳技术优化

Optimization of Two-Dimensional Gel Electrophoresis for Proteome of Chinese Fir Leaves

摘要:

为建立适用于杉木(Cunninghaimia lanceolata)叶片蛋白质组研究的双向电泳技术，对杉木叶片蛋白质的溶解方法、上样量、IEF及SDS－PAGE电泳等关键步骤进行了优化。结果表明，杉木叶片蛋白质主要分布在pH4-7范围；裂解液中含有硫脲（2mmol/L）才能较充分地溶解蛋白，DTT浓度60 mmol/L、上样量1.5mg时得到的图谱分辨率较好且蛋白斑点分布均匀、清晰，拖尾现象明显减少，平衡液Ⅱ中碘代乙酰胺450 mg(15ml)^-1时能提高图谱分辨率；采用与质谱兼容的考马斯亮兰进行染色，得到近700个蛋白点。

Abstract:

A two-dimensional polyareylamide gel electrophoresis(2D-PAGE) system was established for proteomic study of Chinese fir (Cunninghaimia lanceolata) leaves. The result shows that the system containing lysis buffer with 2 mmol/L thiourea for complete dissolution of proteins, DTT 60 mmol/L, loaded proteins 1.5 mg for good protein patterns, equilibration liquid with 450 mg(15 ml)^-1 iodoacetamide for high resolution patterns, colloidal coomassie blue compatible with mass spectrometer. More than 700 protein spots with pH 4-7 were detected.