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

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.

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

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.