Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection |
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Authors: | Tonooka Keiko Kabashima Tsutomu Yamasuji Mutsumi Kai Masaaki |
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Institution: | Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-Machi, Nagasaki 852-8521, Japan. |
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Abstract: | A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins. |
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Keywords: | NF-κB Chemiluminescence detection DNA-binding protein TMPG |
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