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...
| Publié dans: | BioTechniques. - 1991. - 30(2001), 5 vom: 01. Mai, Seite 1044-8, 1050-1 |
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| Auteur principal: | |
| Autres auteurs: | , , |
| Format: | Article |
| Langue: | English |
| Publié: |
2001
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| 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... |
| 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 |
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| Description: | Date Completed 04.12.2001 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |