Kurdistani Siavash K, Grunstein Michael
Department of Biological Chemistry, University of California School of Medicine, Boyer Hall, University of California, Los Angeles, CA 90095, USA.
Methods. 2003 Sep;31(1):90-5. doi: 10.1016/s1046-2023(03)00092-6.
Chromatin immunoprecipitation (ChrIP or ChIP) has commonly been used to map protein-DNA interaction sites at specific genomic loci through use of formaldehyde-induced crosslinking. However, formaldehyde alone has proved inadequate for crosslinking of certain proteins such as the yeast histone deacetylase Rpd3. We report here a modified crosslinking procedure that includes a protein-protein crosslinking agent in addition to formaldehyde. Using this double crosslinking method, we have successfully mapped Rpd3 binding sites in vivo. We also describe the use of ChrIP in combination with DNA microarrays (ChrIP-array) to determine the pattern of Rpd3 binding genomewide. This approach couples the versatility of ChrIP with that of microarrays to identify binding patterns that would otherwise be hidden in a gene-by-gene survey.
染色质免疫沉淀法（ChrIP或ChIP）通常用于通过甲醛诱导的交联来绘制特定基因组位点的蛋白质-DNA相互作用位点。然而，事实证明，仅甲醛不足以交联某些蛋白质，如酵母组蛋白脱乙酰基酶Rpd3。我们在此报告一种改良的交联程序，除甲醛外还包括一种蛋白质-蛋白质交联剂。使用这种双重交联方法，我们已成功在体内绘制了Rpd3结合位点。我们还描述了将ChrIP与DNA微阵列（ChrIP-array）结合使用以确定全基因组Rpd3结合模式的方法。这种方法将ChrIP的多功能性与微阵列的多功能性相结合，以识别在逐个基因调查中可能被隐藏的结合模式。
