A multi-parametric flow cytometric assay to analyze DNA–protein interactions
Name
Arbab-2012-A multi-parametric f.pdf
Size
7.12 MB
Format
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Checksum (MD5)
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Author(s)
Arbab, Mandana
Mahony, Shaun
Cho, Hyunjii
Chick, Joel M.
Rolfe, Philip Alexander
Van Hoff, John Peter
Morris, Viveca W. S.
Gygi, Steven P.
Maas, Richard L.
Gifford, David K.
Date Issued
October 2012
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Citation
Arbab, M. et al. “A Multi-parametric Flow Cytometric Assay to Analyze DNA-protein Interactions.” Nucleic Acids Research 41.2 (2012): e38–e38.
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory
Massachusetts Institute of Technology. School of Science
Terms of Use
Creative Commons Attribution 3.0
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DOI of Published Version
https://doi.org/10.1093/nar/gks1034
