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本研究以薄壳山核桃生长季方块芽接不同发育时期的嫁接为材料,采用不同的蛋白质抽提方法(TCA-丙酮沉淀法,改良TCA-丙酮沉淀法,Tris-HCL浸提法和酚抽提法)、上样量及IPG胶条p H范围等关键因素进行探索与优化,旨在获得薄壳山核桃蛋白的最佳提取方法,并建立适用、高效的薄壳山核桃蛋白质双向凝胶电泳技术体系。Tris-HCL浸提法进行蛋白质的提取效果最佳,可检测到的蛋白质点数量最多约为1 504个,蛋白质中杂质较少,凝胶背景较清晰,基本无横向和竖向条纹,分离出的蛋白质点基本没有拖尾现象。以p H 4~7的IPG胶条、上样量260μg进行双向电泳,蛋白点数量多,分离效果好,基本没有重叠现象,效果最佳。
In this study, we used different protein extraction methods (TCA-acetone precipitation, modified TCA-acetone precipitation, Tris-HCL extraction and phenol extraction Method), sample loading and p H range of IPG strips were explored and optimized in order to obtain the best extraction method for pecan protein from thin shell and to establish a suitable and efficient two-dimensional gel electrophoresis Technology System. Tris-HCL extraction method for the best protein extraction, the maximum number of detectable protein spots is about 1 504, fewer impurities in the protein, the gel background is more clear, basically no horizontal and vertical stripes, isolated The protein spots are basically no tailing phenomenon. Two-dimensional gel electrophoresis was carried out using pGH 4 ~ 7 IPG strips with a loading volume of 260 μg. The number of protein spots was large and the separation effect was good with no overlap, resulting in the best results.