A multi-parametric flow cytometric assay to analyze DNA–protein interactions
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 magnet...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Oxford University Press
2013
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| Online Access: | http://hdl.handle.net/1721.1/77989 https://orcid.org/0000-0003-1709-4034 |
| _version_ | 1826196486815219712 |
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| author | 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. Sherwood, Richard I. |
| author2 | Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory |
| author_facet | Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory 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. Sherwood, Richard I. |
| author_sort | Arbab, Mandana |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T10:27:43Z |
| format | Article |
| id | mit-1721.1/77989 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:27:43Z |
| publishDate | 2013 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | mit-1721.1/779892022-09-30T21:17:49Z A multi-parametric flow cytometric assay to analyze DNA–protein interactions 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. Sherwood, Richard I. Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory Massachusetts Institute of Technology. School of Science Mahony, Shaun Cho, Hyunjii Rolfe, Philip Alexander Gifford, David K. 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. 2013-03-22T21:06:45Z 2013-03-22T21:06:45Z 2012-10 2012-09 Article http://purl.org/eprint/type/JournalArticle 0305-1048 1362-4962 http://hdl.handle.net/1721.1/77989 Arbab, M. et al. “A Multi-parametric Flow Cytometric Assay to Analyze DNA-protein Interactions.” Nucleic Acids Research 41.2 (2012): e38–e38. https://orcid.org/0000-0003-1709-4034 en_US http://dx.doi.org/10.1093/nar/gks1034 Nucleic Acids Research Creative Commons Attribution 3.0 http://creativecommons.org/licenses/by-nc/3.0 application/pdf Oxford University Press Oxford University Press |
| spellingShingle | 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. Sherwood, Richard I. A multi-parametric flow cytometric assay to analyze DNA–protein interactions |
| title | A multi-parametric flow cytometric assay to analyze DNA–protein interactions |
| title_full | A multi-parametric flow cytometric assay to analyze DNA–protein interactions |
| title_fullStr | A multi-parametric flow cytometric assay to analyze DNA–protein interactions |
| title_full_unstemmed | A multi-parametric flow cytometric assay to analyze DNA–protein interactions |
| title_short | A multi-parametric flow cytometric assay to analyze DNA–protein interactions |
| title_sort | multi parametric flow cytometric assay to analyze dna protein interactions |
| url | http://hdl.handle.net/1721.1/77989 https://orcid.org/0000-0003-1709-4034 |
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