Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations.
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.
Lai
J S
JS
Cold Spring Harbor Laboratory, NY 11724.
Herr
W
W
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004996:Ethidium; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051863:Host Cell Factor C1; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007202:Indicators and Reagents; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D051863:Host Cell Factor C1; D007143:Immunoglobulin Heavy Chains; D007202:Indicators and Reagents; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase; D012260:Ribonucleases; D008836:Micrococcal Nuclease; D004996:Ethidium