一种适用于双向电泳的水稻磷酸化蛋白富集方法——酚提取法结合固相金属离子亲和层析法

The Protocol for Phosphoprotein Enrichment in Rice(Oryza sativa L.)——Phenol Extraction Combined with Immobilized Metal Affinity Chromatography

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 very important roles in plant signal transduction and stress responses.Phosphoproteomics has become a big concern for proteomic researchers.Total protein was extracted from rice（Oryza sativa L.Japonica nipponbare） leaves via phenol extraction in this study.Protein mass obtained is up to 3.5% in fresh rice leaves;phosphoprotein was enriched by an immobilized metal affinity chromatography column,which takes a proportion of 6.4% in the total protein.Protein profiles of washing solution,elution solutions and others were approximately exhibited by SDS-PAGE.Based on the results,two-dimensional polyacrylamide gel electrophoresis（2D-PAGE） for total proteins,proteins from peak fraction,non-peak fractions and purified phosphoprotein after enrichment were carried out to better understand the variance among these samples on two dimensional gels.The protocol and procedure described here could dissociate as more as 856 spots per polyacrylamide gel of 7 cm.It is a highly effective way to separate and detecte phosphoproteins.