塔玛亚历山大藻双向电泳蛋白的三种提取方法比较

摘要

[ Objective ] In order to find the best extraction method for proteins of Alexandrium tamarense for two-dimensional electrophoresis (2-DE) analysis. [Methods] Three methods for extracting proteins from A. Tamarense were compared, including trihydroxymethyl aminomethane ( Tris-HCl ) buffer extraction, trichloroacetic acid ( TCA)/acetone precipitation and lysis buffer extraction. Alga was cultivated in normal f/2 media ( control) and supplemented with algicidal substances. Proteins obtained using the best extraction method were separated with 2-DE. Protein-expression differences were identified using matrix-assisted laser desorption/ionization-time of flight ( MALDI-TOF ) mass spectrometry ( MS) . [ Results ] Among the three protein extraction methods, lysis buffer extraction shows the best detection of the number and quality of protein spots with a clear background. Then, the lysis buffer extraction method was successfully applied to profiling protein expression in algicidal substances stress conditions and 14 differential expression proteins were identified using MALDI-TOF/MS. [ Conclusion ] Lysis buffer extraction was the most effective protein extraction method for Alexandrium tamarense.%[目的]比较3种蛋白质提取方法,找到适用于塔玛亚历山大藻蛋白的最佳的提取方法,为后续用双向电泳(2-DE)技术研究不同条件下塔玛亚历山大藻蛋白的差异表达奠定基础.[方法]以塔玛亚历山大藻为研究对象,运用Tris-HCl提取法、TCA沉淀法和lysis buffer提取法分别提取塔玛亚历山大藻蛋白,并通过双向电泳技术,对这3种方法进行了比较分析,筛选出最适于塔玛亚历山大藻的蛋白提取方法.并运用以上得出的方法,以不加杀藻物质的无菌塔玛亚历山大藻为对照,比较分析了塔玛亚历山大藻在加入杀藻物质后的蛋白差异表达状况.[结果]在这3种方法中,lysis buffer提取法得到的蛋白溶解性好,进行双向电泳时,可得到干净的背景、清晰的蛋白点,并且蛋白点的数目较多,酸性蛋白、碱性蛋白、大分子量和小分子量的蛋白均有提出来,蛋白点在胶面上分布均匀.用这种方法初步分析了加入杀藻物质后塔玛亚历山大藻蛋白的差异表达情况,并鉴定出14个与塔玛亚历山大藻生理活动密切相关的蛋白质.[结论¨ysisbuffer提取法获得了最多的蛋白点,双向电泳图谱清晰,适于用来提取塔玛亚历山大藻蛋白.