Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

Forwood, Jade K. and Jans, David A. (2006) Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging. Electrophoresis, 27 16: 3166-3170. doi:10.1002/elps.200500872


Author Forwood, Jade K.
Jans, David A.
Title Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging
Journal name Electrophoresis   Check publisher's open access policy
ISSN 0173-0835
Publication date 2006
Sub-type Article (original research)
DOI 10.1002/elps.200500872
Volume 27
Issue 16
Start page 3166
End page 3170
Total pages 5
Place of publication Weinheim
Publisher Wiley-V C H Verlag Gmbh
Collection year 2006
Language eng
Subject C1
270199 Biochemistry and Cell Biology not elsewhere classified
780105 Biological sciences
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.
Keyword Electrophoretic Mobility Shift Assay
Fluorescence-based Imaging
Protein-dna Interactions
Biochemical Research Methods
Chemistry, Analytical
Mobility Shift Assay
Nuclear-localization Sequence
Import
Site
Sry
Recognition
Complexes
Binding
Trf1
Q-Index Code C1

 
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Created: Wed, 15 Aug 2007, 09:08:26 EST