褐色橘蚜全蛋白双向电泳体系条件优化
Optimization of two-dimensional gel electrophoresis technology system for total protein from brown citrus aphid Toxoptera citricida

为优化有效分离褐色橘蚜Toxoptera citricida全蛋白的双向电泳体系，比较了饱和酚抽提法、三氯乙酸（trichloroacetic acid，TCA）/丙酮沉淀法、直接裂解液法和Tris-HCl法4种蛋白提取方法，以及蛋白上样量、聚焦条件和固相pH梯度（immobilized pH gradient，IPG）胶条对褐色橘蚜全蛋白双向电泳的影响。结果表明，TCA/丙酮沉淀法和直接裂解液法获得的全蛋白浓度分别为4.25、4.03 μg/μL，显著高于饱和酚抽提法和Tris-HCl法，且TCA/丙酮沉淀法所提取的蛋白种类丰富度最高。采用TCA/丙酮沉淀法获得全蛋白，当上样量为100 μg时，所得蛋白质点多、分辨率高、背景清晰，除盐3 h，等电聚焦量45 000 V·h，并选用pH 3~10非线性IPG胶条时，双向电泳图谱中横向拖尾现象少，蛋白点相对清晰，分布均匀。表明所建立的双向电泳体系，结合银染，可从褐色橘蚜全蛋白中识别出1 264个蛋白点，且重复性研究匹配率高，可用于褐色橘蚜蛋白质组学研究。
In order to optimize a two-dimensional electrophoresis (2-DE) system for studying total protein of brown citrus aphid Toxoptera citricida, four different methods of protein extraction including saturated phenol extraction, trichloroacetic acid (TCA)/acetone precipitation, lysate extraction and trihydroxymethyl aminomethane (Tris-HCl) buffer extraction methods were compared. Moreover, the factors including loading amount of protein samples, isoelectric focusing protocols and pH of immobilized pH gradient (IPG) strips were optimized. The results showed that TCA/acetone precipitation and lysate extraction yielded much higher amounts of proteins than other extraction methods, and the protein extraction rate was 4.25 and 4.03 μg/μL, respectively. Furthermore, TCA/acetone precipitation yielded the best richness of proteins. For T. citricida 2-DE, the optimized system included a loading amount of 100 μg protein, desalinated for three hours, with an isoelectric focusing of 45 000 V·h, pH 3-10 nonlinear IPG strip and silver staining, which could produce good background, clear protein spots and few slight streak. This method had detected a total of 1 264 protein spots with a highly reproducible result in the proteomic study of T. citricida.