Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1175395 | Analytical Biochemistry | 2007 | 7 Pages |
Abstract
A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-κB (NF-κB), 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-κB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-κB 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, 3â²,4â²,5â²-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-κB. 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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Keiko Tonooka, Tsutomu Kabashima, Mutsumi Yamasuji, Masaaki Kai,