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目的:建立一套适用于鹿尾腺总蛋白分析的双向电泳体系,为实现通过蛋白质组学研究手段探究鹿尾腺的组织发育及其发挥药理作用的机制奠定基础。方法:使用三氯乙酸(TCA)/丙酮沉淀法、饱和酚抽提法和Trizol法3种方法提取鹿尾腺中总蛋白,采用固相pH梯度胶条进行双向电泳,并对蛋白上样量和等电聚焦参数进行选择和优化,凝胶考马斯亮蓝染色后用PDQuest 8.0软件进行图谱分析。结果:Trizol法得到的总蛋白质纯度最高,能满足双向电泳分析对样品的要求,蛋白质样品在7 cm pH 3～10 IPG胶条上,上样量为300μg,按优化的程序Ⅱ进行双向电泳,可以得到较满意的双向电泳图谱,此时清晰可见蛋白点数多达209个。结论:通过优化双向电泳条件,建立了适合鹿尾腺蛋白质组学研究的双向电泳体系。同时该体系可以为其他类似的动物组织样品制备和双向电泳提供借鉴。
OBJECTIVE: To establish a two-dimensional electrophoresis system suitable for the analysis of the total protein of the canthaal gland, which lays the foundation for exploring the tissue development of the gland’s tail gland and its pharmacological mechanism through proteomics research. Methods: TCA / acetone precipitation method, saturated phenol extraction method and Trizol method were used to extract the total protein in the tail gland. Two-dimensional gel electrophoresis was carried out using the immobilized pH gradient strip. The isoelectric focusing parameters were selected and optimized, and the chromatograms were analyzed by PDQuest 8.0 software after staining with Coomassie brilliant blue. Results: The total protein purity obtained by Trizol method was the highest, which could meet the requirement of two-dimensional electrophoresis analysis. The protein sample was 300μg on 7 cm pH 3 ~ 10 IPG strips. The two- Can be more satisfactory two-dimensional electrophoresis map, clear point of view at this time as many as 209 protein spots. Conclusion: By optimizing the conditions of two-dimensional electrophoresis, a two-dimensional electrophoresis system suitable for the study of proteome of the tail gland was established. At the same time, the system can provide reference for other similar animal tissue sample preparation and two-dimensional electrophoresis.