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建立适用于小菜蛾蛋白质组学研究的样品前处理方法。应用SDS-PAGE电泳比较分析了TCA-丙酮沉淀,Tris-饱和酚抽提,RIPA Lysis Buffer和I-PER Reagent直接裂解4种方法对小菜蛾幼虫总蛋白的抽提质量。结果显示:4种方法对小菜蛾全蛋白的提取率差异比较显著,其中RIPA Lysis Buffer和I-PER Reagent两种直接裂解法蛋白提取率最高,分别为52.06 mg/g和46.16 mg/g;TCA-丙酮沉淀法提取率适中,为34.58 mg/g;Tris-饱和酚抽提法提取率最低,为27.39 mg/g。SDS-PAGE电泳分析表明,Tris-饱和酚抽提法蛋白谱条带分辨率最高,蛋白条带最多,为32条,在17.0 k Da-245 k Da的范围内分布均匀;TCA-丙酮沉淀法蛋白谱带数目为30条,条带清晰,分辨率较高,但缺失部分蛋白;RIPA Lysis Buffer和I-PER Reagent两种直接裂解法蛋白谱条带数目较少,小分子蛋白条带丢失或不明显。根据蛋白提取率和条带的数量综合分析,TCA-丙酮沉淀法适用于小菜蛾蛋白质组学的样品前处理。
A sample preparation method suitable for the study of proteomics of Plutella xylostella was established. SDS-PAGE electrophoresis analysis of TCA-acetone precipitation, Tris-saturated phenol extraction, RIPA Lysis Buffer and I-PER Reagent direct pyrolysis of Plutella xylostella total protein extraction quality. The results showed that there were significant differences among the four methods for the extraction of whole protein of Plutella xylostella, among which RIPA Lysis Buffer and I-PER Reagent had the highest protein extraction rates of 52.06 mg / g and 46.16 mg / g, respectively. TCA - acetone precipitation method was moderate, 34.58 mg / g; Tris-saturated phenol extraction the lowest extraction rate of 27.39 mg / g. SDS-PAGE electrophoresis analysis showed that the resolution of the protein bands of Tris-saturated phenol was the highest, with the largest number of protein bands being 32 and evenly distributed in the range of 17.0 kDa-245 kDa. The TCA-acetone precipitation The number of protein bands was 30, the bands were clear and the resolution was high, but some proteins were missing. The number of protein bands of RIPA Lysis Buffer and I-PER Reagent was less, the bands of small protein were lost or Not obvious. According to the protein extraction rate and the number of bands comprehensive analysis, TCA-acetone precipitation method for the diamondback moth proteomics sample preparation.