Solid phase technology improves coupled gel shift/footprinting analysis

For the analysis of protein-DNA interactions by coupled gel-shift/footprinting, DNA fragments need to be extracted from polyacrylamide gels and subsequently separated on high resolution gels. Due to impurities in the extracted DNA, single nucleotide resolution is frequently not achieved. We now desc...

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Bibliographic Details
Published in:Nucleic acids research 1997-01, Vol.25 (2), p.453-454
Main Authors: Ragnhildstveit, Erlend, Fjose, Anders, Becker, Peter B., Quivy, Jean-Pierre
Format: Article
Language:English
Subjects:
Bacterial Proteins - metabolism
Biotin - analogs & derivatives
Biotin - metabolism
Copper - metabolism
DNA - isolation & purification
DNA - metabolism
DNA Footprinting - methods
DNA-Binding Proteins - metabolism
Electrophoresis, Polyacrylamide Gel
HSP70 Heat-Shock Proteins - genetics
Organometallic Compounds - metabolism
Phenanthrolines - metabolism
Promoter Regions, Genetic - genetics
Protein Binding
Sequence Analysis
Streptavidin
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Description
Summary:For the analysis of protein-DNA interactions by coupled gel-shift/footprinting, DNA fragments need to be extracted from polyacrylamide gels and subsequently separated on high resolution gels. Due to impurities in the extracted DNA, single nucleotide resolution is frequently not achieved. We now describe an improved experimental strategy that employs transient coupling of DNA fragments to a solid support in order to extract DNA of high purity quantitatively, rapidly and reliably. As an example, we describe the application of our protocol to the ‘in-gel footprinting’ by copper phenanthroline. The method should also find application to the chemical interference assays.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/25.2.453