非同位素标记DNA探针和凝胶迁移分析法测定NF-κB激活实验研究  

Detection of NF-κB with non-radioactive labeled DNA probes and gel shift assays

作　　者：牛红心[1] 

机构地区：[1]南方医科大学珠江医院肾内科,广州510282

出　　处：《陕西医学杂志》2011年第3期259-261,279,共4页Shaanxi Medical Journal

基　　金：广东省自然科学基金项目(编号9451051501002540);广东省科技计划项目(编号2010B031600239)

摘　　要：目的:探讨地高辛标记DNA探针和凝胶迁移分析法测定NF-κB激活的优势。方法:用地高辛标记NF-κB基序DNA探针,检测标记效率,探针与核蛋白的结合反应产物经过聚丙烯酰胺凝胶电泳、转膜,高温干燥后,与碱性磷酸酶标记的抗地高辛抗体杂交,加入CSPD检测化学发光强度。结果:按照上述方法标记DNA探针,标记效率较高。样品核蛋白与地高辛标记的NF-κB基序DNA探针共同孵育,两者相互结合,进行凝胶迁移分析,出现高密度的迁移带。在反应体系中额外加入过量未标记的NF-κB基序DNA探针或抗NF-κB/P65或NF-κB/P50抗体后,抑制了两者的结合,迁移带消失,而加入过量未标记的Oct2A基序DNA探针,不能抑制两者结合,迁移带密度无明显降低,证实了核蛋白NF-κB与DNA探针结合的特异性。结论:本方法无放射性污染,无需特殊设备,操作简便、稳定性好、敏感性强,图像清晰、背景低。Objective：To study the advantages of detection of nuclear factor-κB（NF-κB） with digoxingenin-labeled DNA probes and gel shift assays.Methods：DNA probes were labeled with digoxingenin and labeling efficiency was determined.Following the binding reaction of DNA probes and nuclear protein,the complexes were resolved with polyacrylamide gel electrophoresis（PAGE）,and transferred to a positively charged nylon membrane.The membrane was dried,incubated with alkaline phosphatase-conjugated anti-digoxingenin antibody and then detected with CSPD.Results：The labeling efficiency was high.Nuclear protein was combined with digoxigenin-labeled DNA probes and showed a high density shift band after gel shift assays.Competition experiments using unlabeled NF-κB or control Oct2A DNA probes and supershift assays using specific antibodies against P65 and P50 demonstrated the specificity of the DNA protein binding reaction.Conclusion：This method has the advantages of better stability,sensitivity and safety without radioactive contamination and is easy to operate.The pictures are clear with low background.

关 键 词：NF-κB/分析 @凝胶迁移分析实验 DNA探针/利用 

分 类 号：R392[医药卫生—免疫学]