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PMID: 1495986 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 89 ·No. 15 ·1992-08-01 ·Pages 6958-62

Lai JS, Herr W

Abstract

DNA-dependent and DNA-independent associations of DNA-binding proteins are important in transcriptional regulation. The analysis of DNA-independent associations frequently relies on assaying protein interaction in the absence of target DNA sequences. We have found that contaminating DNA in protein preparations can stabilize DNA-dependent associations that may appear DNA-independent. Three cellular proteins of 70, 85, and 110 kDa coimmunoprecipitated with the octamer motif-binding protein Oct-2 because of the presence of contaminating DNA in the cell extracts. In addition, heterodimer formation between Oct-1 (or Oct-2) and Pit-1 during protein-affinity chromatography was stabilized by the contaminating DNA. In both instances, these DNA-dependent protein associations were selectively inhibited by ethidium bromide in the precipitation reaction without any evident effect on DNA-independent protein associations. Thus, ethidium bromide may serve as a simple and general indicator of DNA-dependent and DNA-independent protein associations.

MeSH Terms
Animals Base Sequence Chromatography, Affinity DNA/genetics,isolation & purification,metabolism DNA-Binding Proteins/isolation & purification,metabolism Ethidium Glutathione Transferase/genetics,isolation & purification,metabolism Host Cell Factor C1 Immunoglobulin Heavy Chains/genetics Indicators and Reagents Micrococcal Nuclease Molecular Sequence Data Molecular Weight Octamer Transcription Factor-1 Oligodeoxyribonucleotides Promoter Regions, Genetic Protein Binding Recombinant Fusion Proteins/isolation & purification,metabolism Ribonucleases Transcription Factor Pit-1 Transcription Factors/isolation & purification,metabolism Transfection
Chemicals
DNA-Binding Proteins Host Cell Factor C1 Immunoglobulin Heavy Chains Indicators and Reagents Octamer Transcription Factor-1 Oligodeoxyribonucleotides Recombinant Fusion Proteins Transcription Factor Pit-1 Transcription Factors DNA Glutathione Transferase Ribonucleases Micrococcal Nuclease Ethidium
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Lai J S
Cold Spring Harbor Laboratory, NY 11724.
Herr W
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.89.15.6958
Published
1992-08-01
Pages
6958-62
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC49624
Subset
IM
Grants
NCI NIH HHS · CA13106 · United States
External Links
PubMed source DOI: 10.1073/pnas.89.15.6958 PMC Full Text View journal details
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