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目的:应用蛋白质聚丙烯酰胺凝胶电泳(PAGE)技术筛选建立黄芪蛋白质指纹图谱及蛋白提取方法。方法:采用8种提取方法(稀释4倍的浓缩胶缓冲液,双蒸水,Tris-HCl法,柠檬酸盐法,PVP buffer,改良饱和酚法,TCA-丙酮法,植物蛋白提取试剂盒)提取黄芪药材的水溶性蛋白质,根据PAGE谱带的多少和清晰度优选建立黄芪药材蛋白质指纹图谱的蛋白提取条件。结果:植物蛋白质提取液、稀释4倍的浓缩胶缓冲液和Tris-HCl缓冲液提取的蛋白含量较高,但杂质较多,条带拖尾,部分特征峰不能得到有效分离。TCA-丙酮法、改良饱和酚法、柠檬酸盐缓冲液和PVP buffer提取的蛋白含量较低,谱带数目较少,辨识度低。以双蒸水为提取溶剂时可辨识谱带数目最多,清晰度较好,电泳指纹图谱中各峰的分离度较高。结论:双蒸水直接提取法更适用于建立黄芪药材的蛋白质指纹图谱。
OBJECTIVE: To screen the protein fingerprinting and protein extraction methods of Astragalus by protein polyacrylamide gel electrophoresis (PAGE). Methods: Eight kinds of extraction methods (4 times dilution of concentrated gel buffer, double distilled water, Tris-HCl method, citrate method, PVP buffer, modified saturated phenol method, TCA-acetone method and plant protein extraction kit) The water-soluble protein of Astragalus membranaceus was extracted, and the protein extraction conditions of the protein fingerprinting of Astragalus membranaceus were established according to the number and clarity of PAGE bands. Results: The content of protein extracted by plant protein extract, diluted 4 times concentrated buffer solution and Tris-HCl buffer solution was higher, but more impurities and stripped bands, some of the characteristic peaks could not be effectively separated. TCA-acetone method, modified saturated phenol method, citrate buffer and PVP buffer extracted protein content is lower, the number of bands less, low recognition. When using double-distilled water as the extraction solvent, the number of identifiable bands was the highest, the resolution was better, and the peaks in the electrophoresis fingerprinting had higher resolution. Conclusion: Double distilled water direct extraction method is more suitable for the establishment of the protein fingerprint of Radix Astragali.