摘要
[目的]研究蜂蜜中蛋白质提取的有效方法,优化蜂蜜蛋白电泳条件.[方法]该试验使用丙酮、钨酸钠、硫酸铵、尿素-硫脲4种方法提取蜂蜜中的蛋白并进行比较,然后进行双向电泳分析,对影响双向蛋白电泳的IPG胶条的选择、蛋白质上样量、等电聚焦时间等主要因素进行优化.[结果]试验表明,采用硫脲法提取蜂蜜蛋白获得的蛋白含量最高,硫脲法和丙酮沉淀法获得的蛋白纯度最好,而硫脲法因干扰物质较少、易于裂解适于双向电泳技术,采用硫脲法提取的蜂蜜蛋白选择IPG胶条pH 5～8,蛋白上样量为150μg,等电聚焦时间达到32000 V·h的情况下能充分地聚焦,获得的双向电泳图谱最佳,优化后的洋槐蜜双向电泳图谱上可检测到326个蛋白点,重复性和分辨率均较好.[结论]蜂蜜类含糖样品中使用硫脲法提取蛋白能减少糖类物质干扰.
Abstract
[Objective] To research a effective method for protein extraction from honey, and to optimize the protein electrophoresis conditions of honey.[Method] Protein was extracted from honey by acetone, sodium tungstate, ammonium sulfate and urea-thiourea.Their results were compared and two-dimensional electrophoresis was analyzed.[Result] Protein content was the maximum by thiourea method;protein purity was the highest in thiourea method and acetone precipitation method.Since thiourea method had few interfering substances, and was suitable for two-dimensional electrophoresis technology, we selected thiourea method to extract protein.The IPG adhesive tape was pH 5-8;loading quantity of protein was 150 μg;isoelectric focusing time was 32 000V h.Under these conditions, the optimal two-dimensional electrophoretogram was obtained.A total of 326 protein spots were detected with relatively good repeatability and resolution.[Conclusion] Extracting protein from honey by thiourea method reduces the interference of glucose.
国家科技期刊平台
NSTL
维普
万方数据