Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection.
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.
Tonooka
Keiko
K
Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-Machi, Nagasaki 852-8521, Japan.
Kabashima
Tsutomu
T
Yamasuji
Mutsumi
M
Kai
Masaaki
M
D002457:Cell Extracts; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements / Q000379:methods*; D008851:Micropore Filters; D015394:Molecular Structure; D008970:Molecular Weight; D016328:NF-kappa B / Q000737:chemistry* / Q000302:isolation & purification*; D009698:Nucleoproteins / Q000737:chemistry / Q000302:isolation & purification; D010658:Phenylglyoxal / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification; D014462:Ultrafiltration / Q000295:instrumentation
D002457:Cell Extracts; D007202:Indicators and Reagents; D016328:NF-kappa B; D009698:Nucleoproteins; D014157:Transcription Factors; D010658:Phenylglyoxal