ChIP methodology教程.pdfVIP

PROTOCOL An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants 1,2 ´ 1,3 1 Abdelaty Saleh , Raul Alvarez-Venegas Zoya Avramova 1School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588, USA. 2Center for Integrated Fungal Research (CIFR), Department of Plant Pathology, 3 ´ North Carolina State University, Raleigh, North Carolina 27695, USA. Department of Genetic Engineering, Centro de Investigacion y de Estudios Avanzados, ´ Campus-Guanajuato, Irapuato, C.P. 36821, Mexico. Correspondence should be addressed to A.S. (asaleh@) or Z.A. (zavramova2@). s l Published online 22 May 2008; doi:10.1038/nprot.2008.66 o c o t o Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA–histone r p e interactions in vivo. The procedure includes DNA–histone cross-linking in chromatin, shearing DNA into smaller fragments, r u t immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA a n / sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental m o steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which c . e provides a good yield of high-quality DNA resulting in at least 15 lg of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g r u t of starting tissue material) sufficient to test Z25 genes of interest. T