适用于双向电泳分析的链格孢菌体蛋白提取方法的筛选

为筛选出一套适用于双向电泳分析的链格孢菌蛋白的提取方法,以该菌为研究材料,首先采用TCA-丙酮法、磷酸-TCA-丙酮法、SDS提取法和TCA/丙酮-SDS/酚抽提法分别提取菌丝蛋白质,然后进行蛋白质浓度的测定和单向SDS-PAGE试验,并建立双向电泳体系。结果表明:TCA/丙酮-SDS/酚抽提法所获得的蛋白质浓度为15.9 mg/mL,分别是其他三种方法所得蛋白浓度的2.64、4.61和1.43倍;SDS-PAGE图谱中形成的条带数目最多、丰度最好;所获得的2-DE图谱背景清晰干净,无明显的横纵条纹,分辨率高,可辨别的蛋白质点数为1 138个,是TCA-丙酮法所分离蛋白点数的1.89倍。由此可知,TCA/丙酮-SDS/酚抽提法为链格孢菌蛋白提取的最优方法,该方法所提取的蛋白质适用于双向电泳分析及后续的蛋白质组学研究。

Screening of extraction method of the total protein from Alternariaalternata suitable for two dimensional electrophoresis analysis

In order to screen the extraction methods of total protein from Alternaria alternata suitable for two dimensional electrophoresis analysis, the mycelia proteins in the pathogen were extracted by four methods of TCA acetone, phosphate TCA acetone, SDS and TCA/acetone SDS/phenol, respectively. Furthermore, the protein concentration was analyzed and the separation effect of total protein was detected by SDS PAGE. In addition, a suitable 2-D gel electrophoresis system for mycelia protein was established. The results showed that the protein concentration extracted by the method of TCA/acetone SDS/phenol reached 15.9 mg/mL, which was 2.64, 4.61 and 1.43 times of that extracted by the methods of TCA acetone, phosphate TCA acetone and SDS, respectively. Moreover, the most and clearest protein strips in SDS PAGE gel were obtained by TCA/acetone SDS/phenol extraction method, and the 2-DE map with a distinct background, high resolution, without obvious horizontal and vertical stripes were also obtained. The number of protein points reached 1 138, which is 1.89 times of that isolated by the TCA acetone method. It is suggested that the TCA/acetone SDS/phenol extraction method is the best extraction method of mycelia protein from Alternaria alternata. Meanwhile, the protein extracted by the method is suitable for two dimensional electrophoresis analysis and subsequent proteomic research.