Fiil Berthe Katrine, Qiu Jin-Long, Petersen Klaus, Petersen Morten, Mundy John
Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark.
CSH Protoc. 2008 Sep 1;2008:pdb.prot5049. doi: 10.1101/pdb.prot5049.
INTRODUCTIONTranscriptional reprogramming occurs during development and in response to diverse stimuli and stresses. The isolation and characterization of nuclear proteins, particularly those binding to DNA and chromatin, are therefore important to understanding these processes. Two specific approaches to understanding the function of nuclear proteins involve the characterization of their protein-protein interactions, and of the transcriptional targets of specific transcription factors. Coimmunoprecipitation (co-IP) is a straightforward technique to study in vivo protein-protein interactions, and can identify interacting proteins or protein complexes present in cell extracts. Chromatin immunoprecipitation (ChIP) permits the identification of protein-DNA interactions in pull-down assays using specific antibodies against DNA-binding proteins, such as transcription factors or histone/chromatin-binding proteins. Here, we present detailed protocols for extraction of Arabidopsis seedlings, co-IP of nuclear proteins, and ChIP.
引言
转录重编程发生在发育过程中以及对各种刺激和应激的反应中。因此，核蛋白的分离和表征，尤其是那些与DNA和染色质结合的核蛋白，对于理解这些过程很重要。理解核蛋白功能的两种具体方法包括表征它们的蛋白质-蛋白质相互作用以及特定转录因子的转录靶标。免疫共沉淀（co-IP）是一种研究体内蛋白质-蛋白质相互作用的直接技术，可识别细胞提取物中存在的相互作用蛋白质或蛋白质复合物。染色质免疫沉淀（ChIP）允许在下拉实验中使用针对DNA结合蛋白（如转录因子或组蛋白/染色质结合蛋白）的特异性抗体来鉴定蛋白质-DNA相互作用。在这里，我们提供了拟南芥幼苗提取、核蛋白的免疫共沉淀和染色质免疫沉淀的详细方案。
