Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging.
We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.
Forwood
Jade K
JK
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland, Australia. j.forwood@uq.edu.au
Jans
David A
DA
D000818:Animals; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D024282:Genes, sry; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D011485:Protein Binding; D035321:Telomeric Repeat Binding Protein 1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D015342:DNA Probes; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D035321:Telomeric Repeat Binding Protein 1; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA