|Reference : Use of an Alfexpress DNA Sequencer to Analyze Protein-Nucleic Acid Interactions by Band ...|
|Scientific journals : Article|
|Life sciences : Biochemistry, biophysics & molecular biology|
|Use of an Alfexpress DNA Sequencer to Analyze Protein-Nucleic Acid Interactions by Band Shift Assay|
|Filée, Patrice [Université de Liège - ULg > > Centre d'ingénierie des protéines >]|
|Delmarcelle, Michaël [Université de Liège - ULg > > Centre d'ingénierie des protéines >]|
|Thamm, Iris [Université de Liège - ULg > Département des sciences de la vie > Département des sciences de la vie >]|
|Joris, Bernard [Université de Liège - ULg > > Centre d'ingénierie des protéines >]|
|Yes (verified by ORBi)|
|[en] 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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