用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较  

Comparison of Bovine Whey Protein Preparation Methods for Two-Dimensional Gel Electrophoresis Analysis

作　　者：边艳杰[1] 李庆章[1] 张岚[1] 郭洪波[1] 吕英[1] 

机构地区：[1]乳品科学教育部重点实验室,东北农业大学,哈尔滨150030

出　　处：《中国生物化学与分子生物学报》2011年第5期486-492,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划(973计划,No.2011CB100804);国家科技支撑计划重点项目(No.2008BAK42B05);东北农业大学创新团队计划(No.CXT005-1-1/CXT005-1-2)资助~~

摘　　要：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.Bovine whey contains many kinds of high-abundance proteins and varied interferential impurity, so it＇ s hard to obtain satisfied protein images of bovine whey in two dimensional electrophoresis （2-DE）. Protein extraction from bovine whey is a key step to achieve high-resolution protein separation in 2-DE. Four routine total protein extraction methods were compared in order to determine an optimal one in 2-DE analysis for bovine whey. Bovine whey protein were extracted by direct schizolysis, TCA- acetone, trizol precipitation and 2-D clean up kit. The concentration of total protein was measured by 2- D quant kit, bovine whey protein were separated using SDS-PAGE and 2-DE. The data showed that use of 2-D clean up kit gave maximum protein yield, and the use of trizol gave the lowest. The result of SDS- PAGE showed that use of 2-D clean up kit generated the highest-quality protein among these four methods. The result of 2-DE gels showed that 238 protein spots were detected on the 2-DE gel using 2-D clean up kit, and the gel had almost free of streaking and the best resolution compared with the others; there were 82 protein spots on the gel by using trizol; 118 protein spots appeared on the 2-DE map using TCA-acetone, and the map were contaminated by some interferential impurity; In contrast with above three methods, the image of 2-DE had considerable vertical and horizontal streaking and the lowest resolution using direct schizolysis. It was concluded that the 2-D clean up kit method was much more suitable for the extraction of bovine whey protein in the application of 2-DE, especially for the low- abundance and high molecular weight proteins.

关 键 词：奶牛 乳清蛋白 双向凝胶电泳 制备 

分 类 号：S852[农业科学—基础兽医学] Q503[农业科学—兽医学]