Protocol for Phosphoprotein Enrichment in Rice (Oryza sativa L.) —Phenol Extraction Combined with Immobilized Metal Affinity Chromatography
DENG Xiang-Xiong1), ZHANG Xiao-Qin1), HU Jiang2), XU Xiang-Bin1), GUO Long-Biao2), CHEN Hui-Nan1), YING Qi-Cai1), WANG Hui-Zhong1), XUE Da-Wei1)*
1)College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou310036, China;
2) State Key Laboratory of Rice Biology, National Rice Research Institute of China,Hangzhou310006, China
Abstract: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, nonpeak 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.
邓湘雄,张晓勤,胡江,徐祥彬,郭龙彪,陈慧男,应奇才,王慧中,薛大伟. 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[J]. 中国生物化学与分子生物学报, 2012, 28(11): 1057-1063.
DENG Xiang-Xiong1), ZHANG Xiao-Qin, HU Jiang, XU Xiang-Bin, GUO Long-Biao, CHEN Hui-Nan, YING Qi-Cai, WANG Hui-Zhong, XUE Da-Wei. Protocol for Phosphoprotein Enrichment in Rice (Oryza sativa L.) —Phenol Extraction Combined with Immobilized Metal Affinity Chromatography. Chinese Journal of Biochemistry and Molecular Biol, 2012, 28(11): 1057-1063.
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