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


Autoria(s): Forwood, Jade K.; Jans, David A.
Data(s)

01/01/2006

Resumo

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.

Identificador

http://espace.library.uq.edu.au/view/UQ:80710

Idioma(s)

eng

Publicador

Wiley-V C H Verlag Gmbh

Palavras-Chave #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 #C1 #270199 Biochemistry and Cell Biology not elsewhere classified #780105 Biological sciences
Tipo

Journal Article