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为优化有效分离蚜虫全蛋白的双向电泳体系,以豌豆蚜Acyrthosiphon pisum(Harris)为材料,比较了不同蛋白提取方法(裂解液提取法、三氯乙酸/丙酮沉淀法、饱和酚抽提法)、蛋白上样量、IPG胶条p H梯度等条件对蚜虫全蛋白双向电泳的影响。结果表明:不同的蛋白提取方法所获得的豌豆蚜全蛋白提取率有显著差异,其中以饱和酚抽提法的提取率最高,为5.34±0.26 mg/g,且所提取的蛋白种类丰富度也优于其它2种方法;当蛋白上样量为100μg时所得蛋白点为1 793±36个,高于其它上样量;相同条件下选用11 cm p H 3~10非线性IPG胶条时所得蛋白点为1 803±42个,优于p H 4~7的IPG胶条。表明采用饱和酚抽提法进行提取蛋白,选用11 cm p H 3~10非线性IPG胶条,以蛋白银染方法、分离胶浓度为12.5%进行双向电泳时最适蛋白上样量为100μg,所得可识别蛋白质点最高,且重复性试验匹配率高,可用于豌豆蚜蛋白质组学研究。
In order to optimize and effectively separate the aphid protein, two different methods of protein extraction (lysate extraction, trichloroacetic acid / acetone precipitation, saturated phenol extraction) were compared with Acyrthosiphon pisum (Harris) Protein loading, pG gradient of IPG strips and other conditions on the aphid protein two-dimensional gel electrophoresis. The results showed that the extraction rates of total protein of pea aphids were significantly different with different protein extraction methods, among which the extraction rate was 5.34 ± 0.26 mg / g by saturated phenol extraction method, and the protein variety richness Which is superior to the other two methods. When the amount of protein sample is 100μg, the number of protein spots obtained is 1 793 ± 36, which is higher than that of other samples. When 11 cm p H 3 ~ 10 nonlinear IPG strips are used under the same conditions, The protein spots were 1 803 ± 42, better than IPG strips with p H 4 ~ 7. The results showed that the optimal protein was extracted with saturated phenol extraction method and 11 cm p H 3 ~ 10 non-linear IPG strips were selected. The optimal amount of protein was 100 μg when the concentration of gel was 12.5% The obtained identifiable protein spots are the highest, and the repeatability test has a high matching rate, and can be used for the proteomics research of the pea aphid.