Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays.
Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays.
复制标题
使用染色质免疫沉淀和电泳迁移率变动测定来测定体内和体外蛋白质-DNA 相互作用。
DOI：
10.1007/978-1-59745-277-9_10
复制
发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Imperiale,MichaelJ
中科院分区：
文献类型：
--
作者：
Perez-Romero,Pilar;Imperiale,MichaelJ
病毒生命周期中的许多事件涉及蛋白质与病毒染色体上的限定序列结合。染色质免疫沉淀允许检测特定蛋白质与特定基因组区域的体内相互作用。在这项技术中，活细胞用甲醛处理，以交联相邻的蛋白质和蛋白质DNA分子。与甲醛的交联是可逆的，覆盖的距离很短（2 μ m）;因此，交联的组分非常接近。分离核级分，剪切基因组DNA以将平均DNA片段大小降低至约500 bp。这些核裂解物用于与针对目的蛋白的抗体的免疫沉淀。免疫沉淀后富集与所研究的蛋白质结合的DNA。在交联逆转后，产生的DNA和蛋白质可以独立地进行研究。电泳迁移率变动分析提供了一种快速的方法来研究DNA结合蛋白质的相互作用在体外。该测定法基于蛋白质和DNA的复合物通过非变性聚丙烯酰胺凝胶的迁移速度比游离DNA片段慢的观察结果。通过将纯化的蛋白质或蛋白质的复杂混合物与含有蛋白质结合位点的32 P末端标记的DNA探针孵育来进行测定。在非变性聚丙烯酰胺凝胶上分析反应产物。通过使用特异性和非特异性非放射性标记DNA探针的竞争实验，确定DNA结合蛋白对推定结合位点的特异性。复合物的组分可以用目标蛋白的抗体鉴定。
Many events in the viral life cycle involve protein binding to defined sequences on the viral chromosome. Chromatin immunoprecipitation allows the detection of the in vivo interaction of specific proteins with specific genomic regions. In this technique, living cells are treated with formaldehyde to crosslink neighboring proteinprotein and proteinDNA molecules. The crosslink with formaldehyde is reversible and covers a short distance (2 Å); the components that are crosslinked are therefore in close proximity. Nuclear fractions are isolated, and the genomic DNA is sheared to reduce the average DNA fragment size to around 500 bp. These nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. The DNA bound to the studied protein is enriched after the immunoprecipitation. After reversal of the crosslinking, the resulting DNA and proteins can be independently studied.The electrophoretic mobility shift assay provides a rapid method to study DNA-binding protein interactions in vitro. This assay is based on the observation that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The assay is performed by incubating a purified protein, or a complex mixture of proteins, with a32P end-labeled DNA probe containing the protein-binding site. The reaction products are analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNAbinding protein for the putative binding site is established by competition experiments using specific and nonspecific nonradiolabeled DNA probes. The components of the complexes can be identified with antibodies to the protein of interest.