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231222s2001 xx ||||| 00| ||eng c |
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|a pubmed25n0376.xml
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|a (DE-627)NLM112615511
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|a (NLM)11355340
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|b ger
|c DE-627
|e rakwb
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|a eng
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| 100 |
1 |
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|a Filée, P
|e verfasserin
|4 aut
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|a Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay
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|c 2001
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|a Text
|b txt
|2 rdacontent
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|a ohne Hilfsmittel zu benutzen
|b n
|2 rdamedia
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|a Band
|b nc
|2 rdacarrier
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|a Date Completed 04.12.2001
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|a Date Revised 28.09.2018
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|a published: Print
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|a Citation Status MEDLINE
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|a 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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|a Journal Article
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|a Research Support, Non-U.S. Gov't
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|a Bacterial Proteins
|2 NLM
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|a DNA-Binding Proteins
|2 NLM
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|a Escherichia coli Proteins
|2 NLM
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| 650 |
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|a Lac Repressors
|2 NLM
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| 650 |
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|a Repressor Proteins
|2 NLM
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| 650 |
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|a AmpR protein, Bacteria
|2 NLM
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|a 125267-46-1
|2 NLM
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|a DNA
|2 NLM
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|a 9007-49-2
|2 NLM
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| 650 |
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|a beta-Lactamases
|2 NLM
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| 650 |
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|a EC 3.5.2.6
|2 NLM
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| 700 |
1 |
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|a Delmarcelle, M
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Thamm, I
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Joris, B
|e verfasserin
|4 aut
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| 773 |
0 |
8 |
|i Enthalten in
|t BioTechniques
|d 1991
|g 30(2001), 5 vom: 01. Mai, Seite 1044-8, 1050-1
|w (DE-627)NLM012627046
|x 1940-9818
|7 nnas
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| 773 |
1 |
8 |
|g volume:30
|g year:2001
|g number:5
|g day:01
|g month:05
|g pages:1044-8, 1050-1
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