Enhanced gel mobility shift assay for DNA-binding factors.
Gel mobility shift assays are commonly used to study DNA-binding factors involved in the regulation of constitutive, tissue-specific, and inducible genes. We found that addition of 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate (Chaps, a zwitterionic detergent) at relatively high concentration (2.5% or more) to DNA-binding reactions for four different factors (AP-1, SP1, GATA-1, and interferon alpha-regulated factor ISGF3) assayed in cell extracts greatly enhanced the signal for DNA-protein complexes (up to about 20-fold). The amplified signal for DNA-protein complexes so obtained was (at least in part) due to increased binding efficiency, as revealed by greatly reduced amounts of the free probes in the gels. The binding specificity, however, was not compromised. The fact that DNA-protein complex formation with four different factors was stimulated by Chaps suggests that the enhancing effect of Chaps may be more general and not limited to certain types of DNA-binding factors. The results provide the basis for a highly sensitive assay for DNA-binding factors, which may be useful in several types of studies on such factors. Among other detergents tested, Chapso (another zwitterionic detergent), NP-40, and octylglucopyranoside (nonionic detergents) were found also to enhance the complex formation as tested for AP-1 binding, whereas sodium cholate and deoxycholate showed strong inhibition.
Hassanain
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Hipple Cancer Research Center, Dayton, Ohio 45439.
Dai
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Gupta
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D001483:Base Sequence; D002793:Cholic Acids / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D004586:Electrophoresis; D005347:Fibroblasts / Q000737:chemistry; D005782:Gels / Q000494:pharmacology*; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity
D002793:Cholic Acids; D015342:DNA Probes; D004268:DNA-Binding Proteins; D003902:Detergents; D005782:Gels; D007370:Interferon Type I; D011994:Recombinant Proteins; D004247:DNA; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate