Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method th...

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Détails bibliographiques
Publié dans:BioTechniques. - 1991. - 30(2001), 5 vom: 01. Mai, Seite 1044-8, 1050-1
Auteur principal: Filée, P (Auteur)
Autres auteurs: Delmarcelle, M, Thamm, I, Joris, B
Format: Article
Langue:English
Publié: 2001
Accès à la collection:BioTechniques
Sujets:Journal Article Research Support, Non-U.S. Gov't Bacterial Proteins DNA-Binding Proteins Escherichia coli Proteins Lac Repressors Repressor Proteins AmpR protein, Bacteria 125267-46-1 DNA plus... 9007-49-2 beta-Lactamases EC 3.5.2.6
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Résumé:Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager
Description:Date Completed 04.12.2001
Date Revised 28.09.2018
published: Print
Citation Status MEDLINE
ISSN:1940-9818