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Nat Protoc. 2009;4(3):393-411. doi: 10.1038/nprot.2008.195.

Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.

Author information

1
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

Abstract

Protein-binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA-binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding-site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF-binding specificities at high resolution using such 'all 10-mer' universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day.

PMID:
19265799
PMCID:
PMC2908410
DOI:
10.1038/nprot.2008.195
[Indexed for MEDLINE]
Free PMC Article

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