Chromatin immunoprecipitation and microarray-based analysis of protein location.
Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.
Lee
Tong Ihn
TI
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Johnstone
Sarah E
SE
Young
Richard A
RA
D047369:Chromatin Immunoprecipitation / Q000379:methods*; D019295:Computational Biology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods*; D021381:Protein Transport; D011506:Proteins / Q000032:analysis*; D013010:Sonication
D011506:Proteins