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磷蛋白在植物信号传导和胁迫响应中有着非常重要的作用,磷蛋白质组学研究已经成为蛋白质组学研究领域中备受瞩目的一个部分.本研究用酚提取法以水稻日本晴苗期叶片为材料提取叶片总蛋白,提取率达3.5%;用固相金属离子亲和层析柱纯化富集磷蛋白,得到磷蛋白占总蛋白约6.4%.对过柱洗涤液、不同阶段洗脱液等各个组分进行SDS-PAGE,粗略检测其蛋白含量,并根据单向SDS-PAGE结果对总蛋白、高峰段磷蛋白、非高峰段磷蛋白以及富集后再纯化的总磷蛋白进行双向电泳,比较其中的蛋白差异.本研究提出的方法和程序可在7 cm聚丙烯酰胺凝胶上检测到多达856个磷蛋白,是一种非常有效的磷蛋白富集、纯化和分离鉴定的方法.
Phosphoproteins play a very important role in plant signal transduction and stress response, and Phosphoproteomics research has become a very important part in the field of proteomics.In this study, Extract the total protein of the leaf, the extraction rate was 3.5%; Purification of phosphoprotein by solid-phase metal ion affinity chromatography column to obtain about 6.4% of the total protein of phosphoprotein.On the column washings, different stages of eluate SDS-PAGE was used to detect the content of protein in crude protein. The total protein, peak phosphoprotein, non-peak phosphoprotein and total phosphoprotein purified after enrichment were analyzed by one-way SDS-PAGE. Which are different from each other.The method and procedure proposed in this study can detect up to 856 phosphoproteins on 7 cm polyacrylamide gel, which is a very effective method for the enrichment, purification and identification of phosphoproteins.