曼氏无针乌贼墨囊蛋白质提取及双向电泳条件优化  

Optimization of Protein Isolation and Method of Two-dimensional Electrophoresis from the Ink Sac in Sepiella maindroni

作　　者：詹萍萍[1,2] 王春琳[1,2] 宋微微[1,2] 母昌考[1,2] 李荣华[1,2] 

机构地区：[1]宁波大学海洋学院,浙江宁波315211 [2]宁波大学应用海洋生物教育部重点实验室,浙江宁波315211

出　　处：《宁波大学学报（理工版）》2013年第2期1-6,共6页Journal of Ningbo University：Natural Science and Engineering Edition

基　　金：国家自然科学基金(41176124;41206114);教育部博士点基金(20103305110003)

摘　　要：应用TCA/丙酮沉淀法、裂解液法、裂解液-超速离心法以及层析法4种蛋白质样品制备方法,比较了曼氏无针乌贼（Sepiella maindroni）墨囊蛋白质双向电泳的样品制备效果.在此基础上,比较了不同样品处理方法、不同胶条范围以及不同上样量等对电泳结果的影响,筛选出一套较优的分离墨囊蛋白质的双向电泳条件.研究结果表明：TCA/丙酮沉淀法较适合曼氏无针乌贼墨囊蛋白质双向电泳的样品制备.该方法制备的曼氏无针乌贼墨囊蛋白质样品经双向电泳分离,用固相pH 5~8梯度胶条进行等点聚焦,上样量300μg,电泳结束后硝酸银染色,最终获得了分辨率高、重复性好的双向电泳图谱.经过初步图像分析,检测到（389±19）个蛋白点,背景清晰且蛋白质得率较高.In order to develop an efficient protein extraction method suitable for two-dimensional electrophoresis （2-DE） from the ink sac in Sepiella maindroni, we compared four analytical approaches, which are the precipitation separation of TCA/acetone, the lyolysis buffer, the lyolysis-ultracentrifugation and the column chromatography for total protein extraction. We also compared the effects of different protein extraction methods, different IPG strips and different volumes of sample loaded on the 2-DE. The results show that the TCA/acetone precipitation is the most suitable method for the ink sac protein sample preparation for 2-DE, and the appropriate electrophoresis pattern can be obtained by loading 300 μg protein samples using the method of TCA/acctone precipitation, with IPG strips of pH 5-8 for the first dimensional electrophoresis and staining with silver nitrate. A total of （389±19） protein points are obtained by conducting image analysis. The improved TCA/acetone precipitation can achieve the highest resolution of two dimensional electrophoresis and high yield of protein, and the findings can facilitate the research of proteomics on the ink sac in Sepiella maindroni.

关 键 词：墨囊 蛋白质 双向电泳 

分 类 号：S917.4[农业科学—水产科学]