以白鲢鱼为研究对象,建立并优化其肌原纤维蛋白的双向电泳技术。结果表明,选用固相pH值梯度预制胶条pH4~7,上样量120μg,等电聚焦程序Ⅲ(50V主动水化15h,250V、2h,1000V、2.5h及4000V、3h三段式除盐,8000V、2h和10000V、1.5h两段式升压,10000V聚焦80000V·h,500V保持10h)以及12%的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离胶条件下进行双向电泳,凝胶银染后用PDQuest软件分析,得到具有高分离度的肌原纤维蛋白双向电泳图谱,蛋白点清晰。
A two-dimensional gel electrophoresis (2-DE) system for the separation of myofibrillar proteins from silver carp was established and optimized in this study. The key factors of isoelectric focusing (IEF) and the pH gradient of immobilizedpH gradient (IPG) strip were selected as the main factors affecting 2-DE in this paper. We loaded 120 μg of protein onto the17 cm pH 4-7 IPG strip, and the IEF program III was as follows: 50 V for 15 h, three-stage desalting involving 250 V for 2 h,1 000 V for 2.5 h and 4 000 V for 3 h, 8 000 V for 2 h and 10 000 V for 1.5 h, and 10 000 V for 80 000 V·h and 500 V heldfor 10 h. The gel concentration was 12%. The gel was stained with sliver nitrate, scanned and analyzed using PDQuest software. A high-resolution 2-DE map for myofibrillar proteins from silver carp was obtained finally.