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Nat Protoc. 2006;1(6):2839-55.

ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos.

Author information

1
EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Abstract

This protocol describes a method to detect in vivo associations between proteins and DNA in developing Drosophila embryos. It combines formaldehyde crosslinking and immunoprecipitation of protein-bound sequences with genome-wide analysis using microarrays. After crosslinking, nuclei are enriched using differential centrifugation and the chromatin is sheared by sonication. Antibodies specifically recognizing wild-type protein or, alternatively, a genetically encoded epitope tag are used to enrich for specifically bound DNA sequences. After purification and polymerase chain reaction-based amplification, the samples are fluorescently labeled and hybridized to genomic tiling microarrays. This protocol has been successfully used to study different tissue-specific transcription factors, and is generally applicable to in vivo analysis of any DNA-binding proteins in Drosophila embryos. The full protocol, including the collection of embryos and the collection of raw microarray data, can be completed within 10 days.

PMID:
17406543
DOI:
10.1038/nprot.2006.383
[Indexed for MEDLINE]

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