高通量检测蛋白磷酸化技术研究

Study on a high-throughput technology for measurement of protein phosphorylation

目的研究常规细胞裂解法与AlphaScreen技术结合检测细胞内蛋白磷酸化水平的可行性。方法使用常规配制的细胞裂解缓冲液裂解细胞,分别用传统的Western blot方法和AlphaScreen技术检测在表皮生长因子(EGF)刺激下乳腺癌细胞株MDA-MB-231细胞内AKT磷酸化(p-AKT)水平的变化。结果使用AlphaScreen技术检测到的p-AKT变化趋势和幅度与使用传统Western blot方法检测所得结果一致,同时AlphaScreen技术所得结果更快速。结论常规细胞裂解法所得细胞裂解液可用于AlphaScreen技术检测细胞内蛋白磷酸化水平,该方法使AlphaScreen技术成为一种可以替代Western blot法检测组织和细胞内蛋白激酶活性的高效和灵敏的手段。

Objective To investigate the feasibility of the combination of traditional cell lysis and AlphaScreen technology for measuring protein phosphorylation in cells. Methods MDA-MB-231 cells were stimulated with epidermal growth factor（EGF） for different time and then lysed in traditional lysis buffer.AKT phosphorylation was examined using AlphaScreen technology or Western blot. Results The data obtained by traditional cell lysis/AlphaScreen assay was coordinated with that by Western blot.Conclusion Traditional cell lysis can be utilized with AlphaScreen method for detecting protein phosphorylation in tissues and cells,which is highly effective and sensitive.