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Nucleic Acids Res. 2013 Jan;41(2):e38. doi: 10.1093/nar/gks1034. Epub 2012 Nov 11.

A multi-parametric flow cytometric assay to analyze DNA-protein interactions.

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1
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

Abstract

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA-protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein-DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro.

PMID:
23143268
PMCID:
PMC3554230
DOI:
10.1093/nar/gks1034
[Indexed for MEDLINE]
Free PMC Article
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