Facile determination of DNA-binding nuclear factor-κB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappa B (NF-kappa B), 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-kappa B and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappa B 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-kappa B. 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. (c) 2007 Elsevier Inc. All rights reserved.