高白鲑肌肉蛋白质组双向电泳技术体系的优化

Optimization of Two-dimensional Gel Electrophoresis for Proteome from Coregonus peled Muscle

为建立高白鲑肌肉蛋白质双向电泳分析方法,通过固相pH梯度胶条等电聚焦和SDS-PAGE垂直电泳对蛋白质进行分离,分别考察了蛋白质样品制备方法、上样量、IPG胶条pH值、等电聚焦程序、助凝剂剂量、SDS-PAGE凝胶浓度及染色方法等影响的因素。结果显示,采用丙酮沉淀法制备蛋白质样品,上样量90μg,IPG胶条pH值5~8,一向胶聚焦时间400V16h+1000V2h,平衡时间15min,0.05mL过硫酸铵及2μLTEMED,10%(体积分数)二向分离胶和硝酸银染色时,获得蛋白质分离效果好,分辨率高及横纹少的高白鲑肌肉蛋白质二维电泳图谱,为高白鲑肌肉蛋白质组学的进一步研究奠定了基础。

In order to establish a two-dimensional electrophoresis (2-DE) system for proteomic analysis of Coregonus peled muscle, its muscle proteins were separated using immobilized pH gradient 2-DE. Explored the influence factors such as preparation methods, loading amount, pH of immobilized pH gradients(IPG) gel strips, isoelectric focusing programs, the choice of coagulant aid, the concentration of SDS-PAGE gel and staining methods. The results show that the optimum method for protein samples preparation of Coregonus peled muscle was acetone precipitation. For 2-DE assay, the loading amount was 90 μg, using pH 5-8 IPG gel strips for isoelectric focusing(IEF), time of electrophoresis was 400 V 16 h + 1 000 V 2 h and balanced with 15 min, concentrations of assistant reagents were 0.05 mL ammonium persulfate and 2 μL TEMED, using 10% gel for the second SDS-PAGE and dying the gels by the silver staining. In this condition, we finally got satisfactory 2-DE patterns with good protein separation degree, high repetition and less band, which stands as a valuable resource for further proteomics research of Coregonus peled.