Chromatin Immunoprecipitation (ChIP) in Schizosaccharomyces pombe.
Chromatin immunoprecipitation (ChIP), the cross-linking of chromatin followed by immunoprecipitation with antibodies against a chromatin target, is a key method for measuring association of proteins with a specific genomic region(s). As a negative control, a mock ChIP experiment in which no antibody is added to the immunoprecipitation reaction is included. Enriched DNA fragments from a ChIP experiment can be analyzed in a variety of ways. For semiquantitative analysis, a region of interest can be amplified using standard polymerase chain reaction (PCR) techniques. PCR products are analyzed on agarose (or polyacrylamide) gels and band intensity calculated with a standard imaging software. ChIP enrichment is usually calculated as the ratio of ChIP to input compared with a similar ratio for a reference region not expected to be enriched for that factor. For heterochromatin analysis, housekeeping genes such as act1+are good references. Real-time quantitative PCR (qPCR) can be used for a fully quantitative approach. If a factor is to be mapped across the genome, ChIP DNA can be amplified and labeled for microarray analysis or scrutinized on a next-generation DNA sequencing platform.
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467.
Hugh P
HP
Cam
Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle NE2 4HH, United Kingdom.
Simon
S
Whitehall
D001665:Binding Sites; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis*; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D004268:DNA-Binding Proteins