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Titolo:
Is nitrocellulose filter binding really a universal assay for protein-DNA interactions?
Autore:
Oehler, S; Alex, R; Barker, A;
Indirizzi:
Univ Cologne, Inst Genet, D-50931 Cologne, Germany Univ Cologne Cologne Germany D-50931 nst Genet, D-50931 Cologne, Germany
Titolo Testata:
ANALYTICAL BIOCHEMISTRY
fascicolo: 2, volume: 268, anno: 1999,
pagine: 330 - 336
SICI:
0003-2697(19990315)268:2<330:INFBRA>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
REPRESSOR-OPERATOR INTERACTION; DIFFUSION-DRIVEN MECHANISMS; LAC-REPRESSOR; GEL-ELECTROPHORESIS; NUCLEIC-ACIDS; TRP REPRESSOR; KINETIC MEASUREMENTS; CRYSTAL-STRUCTURE; COMPLEXES; TRANSLOCATION;
Keywords:
filter-binding assay; nitrocellulose binding; mobility shift assay; protein-DNA recognition; Lac repressor;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
52
Recensione:
Indirizzi per estratti:
Indirizzo: Barker, A Univ Cologne, Inst Genet, Weyertal 121, D-50931 Cologne, GermanyUniv Cologne Weyertal 121 Cologne Germany D-50931 ogne, Germany
Citazione:
S. Oehler et al., "Is nitrocellulose filter binding really a universal assay for protein-DNA interactions?", ANALYT BIOC, 268(2), 1999, pp. 330-336

Abstract

The ability to bind to nitrocellulose is commonly accepted as being a universal property of proteins and has been widely used in many different fields of study. This property was first exploited in the study of DNA-binding proteins 30 years ago, in studies involving DNA binding by the lactose repressor (LacR) of Escherichia coli. Termed the filter-binding assay, it remains the quickest and easiest assay available for the study of protein-DNA interactions. However, the exact mechanism by which proteins bind to nitrocellulose remains uncertain. Given the supposedly universal nature of the interaction, we were surprised to notice that certain LacR variants were completely unable to bind simultaneously to DNA containing a single lac operator and nitrocellulose. Investigation of this loss of binding suggests that LacRrequires a protein region that is both hydrophobic in nature and more or less unstructured, in order to bind to both nitrocellulose and DNA. In the case of wild-type, tetrameric LacR, the DNA-recognition domain that is not bound to DNA suffices. Dimeric LacR variants will only bind if they have certain C-terminal extensions. These experiments sound a cautionary note for the use of filter binding as an assay of choice,particularly in applicationsinvolving screening for the DNA-binding site of putative DNA-binding proteins. (C) 1999 Academic Press.

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Documento generato il 24/09/20 alle ore 21:33:00