黄芪蛋白提取方法的比较

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。

Protein Extraction Methods of Astragali Radix

Objective: To select the protein extraction protocols for establishing biological fingerprint of Astragali Radix by using PAGE technology. Method: The water soluble protein of Astragali Radix was extracted by 8 different methods (concentrated gel buffer diluted by 4 times, distilled water, Tris-HCl method, the citrate method, PVP buffer, the modified saturated phenol method, TCA-acetone method and protein extract solution) and the PAGE experiment was conducted. The optimal protein extraction protocols for establishing biological fingerprint of Astragali Radix were selected according to the number and the definition of the protein bands. Result: The content of protein extracted by protein extract solution, concentrated gel buffer diluted by 4 times and Tris-HCl buffer was higher, but it showed a higher content of impurities, a dn some characteristic peaks cannot effectively isolated;the content of bands of protein extracted by TCA-acetone method, the modified saturated phenol method, PVP buffer and citrate buffer were lower, with a smaller number of bands and lower definition;bands of protein extracted by distilled water were most numerous and stabler, with a higher separation among peaks in electrophoresis fingerprint. Conclusion: The protein solution extracted with distilled water is more suitable for establishing the protein fingerprint of Astragali Radix.