Department of Biology/Biochemistry, University of Fribourg, Fribourg, Switzerland.
Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
Methods Mol Biol. 2021;2130:115-125. doi: 10.1007/978-1-0716-0381-9_9.
Understanding the binding of regulatory proteins to their cognate genomic sites is an important step in deciphering transcriptional networks such as the circadian oscillator. Chromatin immunoprecipitation (ChIP) enables the detection and temporal analysis of such binding events in vivo. Here, we describe the individual steps from the generation of formaldehyde-cross-linked chromatin from mouse liver nuclei, fragmentation thereof, immunoprecipitation, reversal of cross-links, fragment cleanup, and detection of binding sites by real-time PCR. Depending on the quality of the employed antibody, a clear enrichment signal over the background is expected with a resolution of about 500-800 base pairs around the selected primer-probe pair.
理解调节蛋白与其同源基因组位点的结合是破译转录网络（如生物钟振荡器）的重要步骤。染色质免疫沉淀（ChIP）可用于在体内检测和分析这种结合事件。在这里，我们描述了从鼠肝细胞核中生成甲醛交联染色质、片段化、免疫沉淀、交联逆转、片段清洗以及通过实时 PCR 检测结合位点的各个步骤。根据所使用的抗体的质量，预计在选定的引物-探针对周围约 500-800 个碱基处会出现明显高于背景的富集信号。
