摘要
目的应用奥德赛(Odyssey)近红外双荧光系统筛选去甲基化酶KDM3A去磷酸化的磷酸酯酶。方法使用磷酸酯酶干扰质粒转染HEK293T细胞,使用磷酸化的KDM3A抗体对细胞进行杂交,近红外荧光染料DRAQ5标记细胞核,利用奥德赛(Odyssey)近红外双荧光系统对细胞进行荧光扫描,荧光强度的比值可代表细胞内相对KDM3A磷酸化水平,进而筛选出相关的磷酸酯酶。结果磷酸酯酶广谱抑制剂冈崎酸OA处理转染后的细胞,近红外双荧光扫描系统可以检测到KDM3A的磷酸化,初步筛选出4个可能参与KDM3A去磷酸化的磷酸酯酶亚基。结论近红外双荧光系统可以应用于磷酸酯酶的初步筛选。
Objective To determine the phosphates of histone demethylase KDM3A with the application of Odyssey dual - infrared fluorescence system. Methods HEK293T cells had been transfected with phosphates interfere plasmids. After incubating with phospho- rylation - KDM3A antibody and infrared fluorescent nuclei dyes DRAQ5, HEK293T cells were scanned with Odyssey dual - infrared fluo- rescence scan system. The ratio of fluorescence intensity could represent the relative KDM3A phosphorylation level in vivo. Results Oka- zaki acid （OA） , the broad - spectrum inhibitor of phosphates, was applied to treat the cells. The signals were detected for phosphorylated KDM3A by dual- infrared fluorescence scan system. Four phosphate subunlts that may participate in the dephosphorylation of KDM3A were determined by Odyssey dual - infrared fluorescence scan system. Conclusion Odyssey dual - infrared fluorescence scan system was able to be applied in the screening for protein phosphotases.
出处
《医学研究杂志》
2014年第5期52-54,共3页
Journal of Medical Research
基金
教育部博士点基金资助项目(20111106110024)
