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

Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Protocols Springer Journals

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

Berger, Michael F; Bulyk, Martha L
Nature Protocols , Volume 4 (3) – Mar 5, 2009
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References (66)

Publisher
Springer Journals
Copyright
Copyright © 2009 by Nature Publishing Group
Subject
Life Sciences; Life Sciences, general; Biological Techniques; Analytical Chemistry; Microarrays; Computational Biology/Bioinformatics; Organic Chemistry
ISSN
1754-2189
eISSN
1750-2799
DOI
10.1038/nprot.2008.195
Publisher site
See Article on Publisher Site

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.

Journal

Nature Protocols – Springer Journals

Published: Mar 5, 2009

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