A Rapid and High-Throughput Quantitation Assay of the Nuclear Factor κB Activity Using Fluorescence Correlation Spectroscopy in the Setting of Clinical Laboratories

Abstract

BackgroundTranscription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-κB activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-κB to fluorescent-labeled DNA probes, which contain NF-κB binding sites.MethodsNon-fluorescent competitive probes are employed to normalize the influence of the viscosity of the nuclear extracts between samples and to eliminate the influence of nonspecific binding of the fluorescent probes. To confirm accurate quantitation, human recombinant NF-κB p50 was mixed into U937 cell nuclear extracts, and the binding fraction of the fluorescent probes to NF-κB in the mixture was calculated for quantitation. To evaluate whether this method can be applied to measure the NF-κB activity in human lymphocytes, the NF-κB activity levels of systemic inflammatory response syndrome patients during perioperative periods were measured.ResultsThe percentage recovery was 88.9%. The coefficients of variation of the intra-assay were approximately 10%. NF-κB activity levels during the perioperative period can were successfully measured. The assay time for the FCS measurement was within 20 minutes.ConclusionsThis assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.

Affinity‐based high‐resolution analysis of DNA binding by VASCULAR‐RELATED NAC‐DOMAIN7 via fluorescence correlation spectroscopy

“…A transient reporter assay and EMSA showed that VND7 binds to the 53‐bp X1E1 sequence of the XCP1 promoter (from −148 to −96 bp; Yamaguchi et al ., ; Figure S1b). In this study, we first aimed to define which nucleotides within the X1E1 sequence are critical for interaction with VND7 by using FCS, which allowed us to determine the status of the interaction between DNA and proteins quantitatively (Kinjo and Rigler, ; Wölcke et al ., ; Kobayashi et al ., ; Octobre et al ., ; Veprintsev and Fersht, ; Harada et al ., ). For the FCS analysis, we prepared a maltose‐binding protein (MBP)‐tagged C‐terminally truncated VND7 protein containing the whole NAC domain (amino acid residues 1–161; MBP‐VND7 1–161 ; Yamaguchi et al ., , ; Endo et al ., ) and the 138‐bp promoter fragment of XCP1 (from −233 to −96 bp) labeled with a fluorescent dye, 5(6)‐carboxytetramethylrhodamine (TAMRA) (TAMRA‐ XCP1 pro).…”

“…However, SPR is considered to be suitable for the detailed analysis of only limited numbers of protein and/or DNA samples, because the molecules need to be immobilized on the sensor chips (Helwa and Hoheisel, ). In contrast, FCS not only is a quantitative method for analyzing the affinity between transcription factors and DNAs, but also has the advantage in analyzing a large number of samples because the protein and DNA molecules are simply mixed in a solution at small scale (Kinjo and Rigler, ; Wölcke et al ., ; Kobayashi et al ., ; Octobre et al ., ; Veprintsev and Fersht, ; Harada et al ., ). Therefore, a variety of different sets of competitor fragments can be used for FCS, which is thus suitable for high‐resolution analysis of cis ‐acting sequences responsible for binding to the specific proteins.…”

“…Here, we showed that FCS is a useful tool for evaluating the contribution of each nucleotide within the responsible region to the binding affinities. FCS is unsuitable for analyzing binding between molecules with small molecular weight changes after binding (Kinjo and Rigler, ; Wölcke et al ., ; Kobayashi et al ., ; Octobre et al ., ; Veprintsev and Fersht, ; Harada et al ., ), but the molecular weight changes in the experiments testing interactions between transcription factors and DNA fragments are typically large enough to be accessible to FCS.…”

“…The dissociation constant between TAMRA‐labeled DNA and MBP‐tagged protein ( K d value) was calculated from the Hill equation (Hill, , ). A fixed concentration (5 n m ) of TAMRA‐labeled DNA was incubated with different concentrations of the MBP‐tagged protein (from 0.01 to 0.161 μ m ), and then the binding fraction ( α ), which denotes the ratio of the labeled DNA binding to the protein to the total labeled DNA (Harada et al ., ), at each protein concentration was obtained. The values of α at each protein concentration divided by the maximum values of α were plotted against the concentrations of each protein, which were fitted to the Hill equation (below) to calculate the K d values.where y and y 0 are the values of α at each protein concentration and a maximum value of α , respectively; [E] is the protein concentration; and n is the Hill coefficient.…”

“…As the diffusion time of a labeled molecule depends on its molecular weight, larger molecules move slowly and exhibit a longer diffusion time while smaller molecules move fast and thus exhibit a shorter diffusion time (Haustein and Schwille, ), interactions between the labeled molecules and other molecules can be evaluated in accordance with the changes in diffusion time (Figure S1a). Moreover, FCS can be used for competition assays using unlabeled DNA molecules as competitors (Wölcke et al ., ; Kobayashi et al ., ; Veprintsev and Fersht, ; Harada et al ., ). The results of such competition assays can be used in kinetic analyses as a means to calculate binding affinities (Wölcke et al ., ; Veprintsev and Fersht, ).…”