Advancing Epigenetics Towards Systems Biology

Chromatin immunoprecipitation protocol for Saccharomyces cerevisiae (Prot 27)

Haico van Attikum & Jennifer Cobb

Introduction

ChIP (chromatin immunoprecipitation) is a powerful tool that allows one to determine whether and where a protein or protein modification is associated with chromatin in vivo. The technique starts with a formaldehyde treatment of cells to crosslink protein-protein and protein-DNA complexes. After cross-linking, the cells are lysed and crude extracts are sonicated to shear the DNA. Proteins together with crosslinked DNA are immunoprecipitated. Protein-DNA crosslinks in the immunoprecipitate and input (non-immunoprecipitated whole cell extract) are then reversed and the DNA fragments are purified. Real-time quantitative PCR can then be used to amplify the region where either a protein or protein modification is present. DNA fragments of this genomic locus should be enriched in the immunoprecipitate compared to that in the input (which represents all portions of the genome). This protocol has been successfully used in the Gasser lab to study proteins at replication forks and chromosomal DNA double-strand breaks (DSBs) in budding yeast (Saccharomyces cerevisiae) (Cobb et al., 2003; van Attikum et al., 2004).

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Haico van Attikum & Jennifer Cobb

Friedrich Miescher Institute for Biomedical Research - Maulbeerstrasse 66 - 4058 Basel, Switzerland

Haico van Attikum & Jennifer Cobb