Advancing Epigenetics Towards Systems Biology

Chromatin Immunoprecipitation Protocol for Histone Modification & Chromatin Associated Proteins (Prot 3)

Roderick O'Sullivan & Joost Martens

Introduction

Chromatin Immunoprecipitation (ChIP) experiments are routinely performed in many laboratories around the world to examine the occupancy of proteins or chromatin modifications over particular stretches of the genome. Briefly, chromatin extracts are prepared following fixation with formaldehyde. This bulk chromatin is then sheared into fragments of useful sizes. Aliquots are then incubated with an antibody of choice and immuno-complexed DNA is purified and analyzed, invariably by PCR. However, much discussion remains as to how best these types of experiments are performed, in particular, the method of chromatin lysate preparation, sonication efficiency, antibody concentration and washing of the immuno-complexes. In this protocol, we will describe the method used in our laboratory. This protocol has been successfully used in experiments with Mouse Embryonic Stem (ES) cells, Embryonic Fibroblasts (MEF), Trophoblast Stem (TS) cells, B-lymphocytes and hepatocytes, as well as with U2OS, 293, Hela, T98G cells from human. Nonetheless, we would urge each investigator to expend much effort in standardizing the method for their specific experimental system.

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Roderick O'Sullivan & Joost Martens

Jenuwein Lab - Research Institute of Molecular Pathology (IMP) - Dr. Bohr-Gasse 7 - A1030 Vienna, Austria

Roderick O'Sullivan & Joost Martens