Quantitative analysis of DNA‐protein interactions using double‐labeled native gel electrophoresis and fluorescence‐based imaging
- 16 August 2006
- journal article
- Published by Wiley in Electrophoresis
- Vol. 27 (16) , 3166-3170
Abstract
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.Keywords
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