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目的优化双向凝胶电泳的实验步骤,总结一套适用脾脏组织的双向凝胶电泳技术。方法对双向电泳实验中的关键环节:蛋白沉淀方法;胶条PH的选择;聚焦条件等进行研究与优化,考马斯亮蓝染色后进行凝胶图像比较。结果 Cleaning up沉淀蛋白,聚焦电压达到11 000伏小时(110k vhs),可以得到多达1000个蛋白点的2D gel(two-dimensional polyacrylamide gel)。结论建立了脾脏双向电泳的具体方法,得到分辨率高重复性好的2D gel,为脾脏的蛋白质组学研究奠定了基础。
Objective To optimize the experimental procedure of two-dimensional gel electrophoresis and to summarize a two-dimensional gel electrophoresis technique suitable for spleen tissue. Methods The key steps in two-dimensional electrophoresis experiments were: protein precipitation method, selection of strip pH, focusing conditions and so on. The gel images were compared after staining with Coomassie Brilliant Blue. Results Cleaning up Precipitated protein, 2D gel (two-dimensional polyacrylamide gel) with up to 1000 protein spots was obtained with a focused voltage of 11 000 volts (110k vhs). Conclusion The specific method of two dimensional gel electrophoresis in spleen was established. 2D gel with high resolution and good repeatability was established, which laid the foundation for proteomics research of spleen.