燕窝蛋白质制备及双向电泳分离条件的研究

Preliminary research of sample preparation and separation for two-dimensional electrophoresis of proteins from edible bird nest

样品制备是双向电泳成败的关键,为此以市场上购买的燕盏为材料,对燕窝蛋白质样品制备中的蛋白质沉淀及沉淀洗涤等条件进行了比较,发现常用的蛋白质沉淀方法包括丙酮沉淀法、三氯乙酸沉淀法、三氯乙酸/丙酮沉淀法及酚抽提法中,以丙酮沉淀法制备样品SDS凝胶电泳后获得的蛋白质谱带最为丰富,80%丙酮去除蛋白质沉淀中杂质的效果优于丙酮。双向电泳分析发现燕窝蛋白质主要是酸性蛋白质,采用窄范围pH3～5.6的非线性胶条,对其进行了较好的分离。双向电泳分离体系的建立,为燕窝蛋白质图谱库的构建及相关研究提供了条件。

Protein preparation was the key step for two-dimensional electrophoresis,so the sample preparation of proteins from edible bird nest including protein precipitation methods and washing methods were compared. The effect of different protein precipitation methods based on acetone,Trichloroacetic acid（TCA） ,TCA/acetone and Tris-saturated phenol method,was analyzed. The results showed that the acetone precipitation method was the best one resulting in the most protein bands and the best resolution in SDS-polyacrylamide gel electrophorsis（PAGE） . It was also found that the effect of 80 percent acetone to get rid of impurities especially salt ions from protein precipitation was better than 100 percent acetone. The two-dimensional electrophoresis gels of acetone precipitation showed proteins in edible bird nest mainly distributed in the acid end. Using pH3～5.6 IPG strips has better separation results. The establishment of methods to separate proteins from edible bird nest with two-dimensional electrophoresis provided conditions for constructions of protein database and related researches.