RRC ID 42207
Author Kobayashi T, Okamoto N, Sawasaki T, Endo Y.
Title Detection of protein-DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy.
Journal Anal Biochem
Abstract Fluorescence correlation spectroscopy (FCS) is a methodology to examine directly the translational diffusion of individual fluorescence-labeled molecules in solutions. Recent studies using FCS have quantified various bimolecular reactions without any need for amplification. To evaluate further the applicability of FCS, we studied the specific binding between proteins and DNA in crude biological samples. Using an automated FCS system that was recently developed in our laboratories and is capable of distinguishing two or more molecular species in a multicomponent analysis, we detected the binding of two representative transcription factors, activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), in nuclear extracts of HeLa cells quantitatively with each sequence-specific DNA. The binding rates of these specific interactions were markedly augmented when cells were treated with tumor necrosis factor alpha which is known to activate both AP-1 and NF-kappaB. We also observed the pyrrolidine-dithiocarbamate-induced reciprocal regulation of these transcription factors. These results indicated that FCS is a useful tool for the analysis of complex interactions of transcription factors with DNA even in crude cellular extracts, suggesting that it is a powerful methodology for the study of a wide variety of molecular events under various experimental conditions.
Volume 332(1)
Pages 58-66
Published 2004-9-1
DOI 10.1016/j.ab.2004.05.053
PII S0003269704004920
PMID 15301949
MeSH DNA / metabolism* HeLa Cells Humans JNK Mitogen-Activated Protein Kinases / metabolism Luminescent Measurements / methods* NF-kappa B / metabolism Protein Binding / physiology Proteins / metabolism* Spectrometry, Fluorescence / methods Transcription Factor AP-1 / metabolism Tumor Necrosis Factor-alpha / metabolism
IF 2.507
Times Cited 17
WOS Category CHEMISTRY, ANALYTICAL BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Human and Animal Cells