为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2-D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.
Prolactin(PRL) is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be necessary for high levels of milk protein expression, the mechanism by which the synthesis of milk proteins is stimulated at the transcript level is less known. A major modification in the transcript level is protein phosphorylation. To gain additional insights into the molecular mechanisms at the transcript level underlying PRL action on the dairy cow mammary epithelial cells(DCMECs), nuclear phosphoproteins whose expression distinguishes proliferating regulated by PRL in DCMECs were identified. A phosphoprotein-enriched fraction from nuclear proteins was obtained by affinity chromatography, and a two-dimensional gel electrophoresis(2-DE) and matrix assisted laser desorption/ionization time of matrix-assisted laser desorption/ionization/time of flight mass spectrometry(MALDI-TOF MS)were used to identify the changes of nuclear phosphoproteins in DCMECs treated with prolactin. Seven proteins displaying≥2-fold difference in abundance upon PRL treatment in DCMECs were identified by MALDI-TOF MS. The protein-GARS(GlyRS), which belonged to the class-II aminoacyl-tRNA synthetase family, played a global role in the milk protein synthesis. SERPINH1(Heat shock protein 47), which was the first heat shock protein found to be a member of the serpin superfamily, regulated physiologic functions,such as complement activation, programmed cell death, and inflammatory processes. PRDX3, which belonged to a family of antioxidant enzymes, played an important role in scavenging intracellular reactive oxygen species(ROS). ACTR1A, belonged to the actin family, which was associated with transport of p53 to the nucleus. Annexin A2, a Ca2+-dependent phospholipid-binding protein,maintained the viability and cell cycle regulation of DCMECs. PSMB2 and PSMD10, which belonged to ubiquitin-proteasome system, were involved in several c
