坛紫菜叶状体蛋白质双向电泳样品制备方法的比较

Comparison of protein preparation methods of Porphyra haitanensis thalli for two-dimensional electrophoresis

蛋白质样品的制备是双向电泳分析的关键。为探索适用于坛紫菜叶状体蛋白质双向电泳样品的制备方法,实验对3种常用植物蛋白质分离方法进行了优化和改进,并分别同3种蛋白质裂解液联用,比较了9种方法的总蛋白质得率及双向电泳图谱效果。结果发现,采用三氯乙酸/丙酮沉淀法与裂解液C(7 mol/L尿素,2 mol/L硫脲,2%CHAPS,2%TritonX-100,2%IPG Buffer pH 3～10,65 mmol/L DTT)联用的方法,坛紫菜叶状体的蛋白质得率最高,且双向电泳图谱显示此法获得的蛋白质点数最多,蛋白点形状规则,水平和垂直拖尾情况较轻,图像清晰。实验结果还发现,在进行双向电泳分析时,采用pH 4～7的胶条可以使坛紫菜叶状体蛋白质得到更为有效的分离。

Protein preparation is a key step in two-dimensional electrophoresis（2-DE）analysis.In order to establish a suitable protein preparation method for Porphyra haitanensis thalli,three sample preparation methods of plant,trichloroacetic acid/acetone precipitation（TCA/ACE）method,urea/thiourea extraction（UREA/THI）method and phenol extraction methanol/ammonium acetate precipitation method,and three protein lysis buffers were compared.The results showed that the optimal method of proteins preparation of P.haitanensis thalli was extracted using TCA/ACE method and dissolved with the lysis buffer C（7 mol/L urea,2 mol/L thiourea,2%CHAPS,2%TritonX-100,2% IPG Buffer pH 3-10,65 mmol/L DTT）.Using this method,the yield was the highest and 2-DE map has the most spots,clear backgrounds,high resolution and great repeatability.At the same time,we also found that using the IPG strips（pH 4-7）in 2-DE analysis can extract the protein of P.haitanensis thalli better than IPG strips（pH 3-10）.