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 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.
Filée
P
P
Université de Liège, Liège, Belgium.
Delmarcelle
M
M
Thamm
I
I
Joris
B
B
D001322:Autoanalysis; D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D016971:Citrobacter freundii / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; C071526:AmpR protein, Bacteria; D004247:DNA; D001618:beta-Lactamases