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 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.
Ragnhildstveit
E
E
Department of Biochemistry and Molecular Biology, University of Bergen, Aarstadv, 19 N-5009 Bergen, Norway.
Fjose
A
A
Becker
P B
PB
Quivy
J P
JP
D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D009942:Organometallic Compounds / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017421:Sequence Analysis; D019809:Streptavidin
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D009942:Organometallic Compounds; D010618:Phenanthrolines; C078638:copper-neocuproine complex; D001710:Biotin; D003300:Copper; D004247:DNA; D019809:Streptavidin