解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (6 ): 865-868.doi: 10.3969/j.issn.0529-1356.2013.06.023

• 细胞和分子生物学 • 上一篇    下一篇

凝胶电泳蛋白质组学蛋白样品纯化方法比较

卜旺雨1 齐义军1*  魏华1 张娟1 马瑾1 王明1 马远方1   

  1. 1. 河南大学医学院细胞与分子免疫学重点实验室，河南 开封 475004; 2. 河南大学淮河医院内镜室，河南 开封 475000
  • 收稿日期:2013-02-21 修回日期:2013-03-31 出版日期:2013-12-06 发布日期:2013-12-06
  • 通讯作者: 齐义军 E-mail:qiqiyijun@gmail.com
  • 基金资助:

    国家自然科学基金重点资助项目

Comparison of protein preparation methods for Gel-based proteomics

BU Wang-yu1 QI Yi-jun 1* WEI Hua2 ZHANG Juan1 MA Jin1 WANG Ming1 MA Yuan-fang1   

  1. 1. Key Laboratory of Cellular and Molecular Immunology, College of Medicine He’nan Kaifeng 475004, China;2. Department of endoscopy, Huaihe Hospital, Henan University, He’nan Kaifeng 475000, China
  • Received:2013-02-21 Revised:2013-03-31 Online:2013-12-06 Published:2013-12-06
  • Contact: QI Yi-jun E-mail:qiqiyijun@gmail.com

摘要：

目的 探讨超滤法和7种不同沉淀方法纯化含PBS的蛋白样品对蛋白凝胶电泳的影响。方法 应用超滤法和7种沉淀方法［包括硫酸铵、氯仿甲醇、酸化丙酮甲醇、乙醇、丙酮、三氯乙酸（TCA）、TCA/丙酮］纯化PBS稀释的混合食管癌组织蛋白（10例），二喹啉甲酸（BCA）、Bradford及2D法测定蛋白浓度并进行一维和二维聚丙烯酰胺凝胶蛋白电泳，Image Master软件分析二维电泳图谱蛋白点。 结果 Bradford法和2D法适用于二维电泳水化液溶解的蛋白样品浓度测定。超滤法和硫酸铵沉淀法的蛋白回收率分别为40%和4%，其他方法的蛋白回收率约为22%。丙酮沉淀法纯化蛋白的二维电泳图谱蛋白点边界清晰、分辨率高且数目最多；其次是乙醇沉淀和氯仿甲醇沉淀法；再其次是超滤法，该法纯化蛋白的二维电泳图谱蛋白点表现不同程度的弥散；酸化丙酮甲醇沉淀蛋白的二维电泳图谱中蛋白点丢失最多。 结论 丙酮沉淀法适用于原始浓度高、体积小的蛋白样品除盐及浓缩，超滤法是原始体积较大、浓度较低蛋白样品浓缩纯化的首选方法。

关键词: 食管, 蛋白样品制备, 蛋白质组, 蛋白沉淀, 超滤, 二维电泳

Abstract:

Objective To evaluate the efficiency of protein preparation for human esophageal tissue protein diluted in PBS by ultracentrifugation and 7 protocols for protein precipitation. Methods Eight protocols, which included ultracentrifugation and 7 protocols for protein precipitation ［ammonium sulfate, chloroform/methanol, acidified acetone/methanol, ethanol, acetone, trichloroacetic acid (TCA), TCA/acetone］, were used to extract PBS-diluted mixed protein samples derived from human esophageal cancer tissue (10 cases). Protein concentration was determined by bicinchoninic acid(BCA), Bradford and 2D quantification kits. The recovered protein diluted in 2D rehydration buffer was separated either by one-dimensinal mini-gel electrophoresis or two-dimensional gel electrophoresis (2D) followed by ImageMaster software analysis. Results Protein quantification protocols by Bradford and 2D were compatible with 2D rehydration buffer. With the exception of the protein recovery rates for the ultracentrifugation and ammonium sulphate precipitation which were 40% and 4%, respectively, the protein recovery rates for remained methods were 22%. In view of protein spot boundary, number and resolving ability, aectone-precipitated protein produced the best quality of 2D images, followed by precipitation protocols of ethanol and chloroform/methanol, and ultracentrifugation. Acidified acetone/methanol protocol produced the greatest protein spot lost. Conclusion Acetone precipitation protocol is feasible with protein samples with high concentration and small volume, while ultracentrifugation is for other protein samples.

Key words: Esophagus, Protein preparation, Proteomics, Protein precipitation, Ultracentrifugation, Two-dimensional electrophoresis