NF-kB-p65 protein:DNA binding assay

Figure 1: Schematic of NF-k B competition binding assay.

The transcription factor, NF-kB has received extensive interest over the past decade because of its pivotal role in cellular processes, such as inflammation and apoptosis(1, 2). We have examined the interaction between the p65 subunit of NF-kB and a 19 base pair double stranded (ds) DNA sequence containing the 10 base pair recognition site for NF-kB. The FRET assay comprises a Cy™3 labeled anti-GST antibody which specifically binds to a p65-GST tagged protein, followed by interaction with NF-kB dsDNA 5’ end-labeled on the coding strand with Cy5.

The FRET signal generated when the three components are bound together can be disrupted with an excess of unlabeled NF-kB-specific dsDNA. (Figure 1).

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Methods

Assays were carried out in black opaque 384-well (maximum working volume 80ml) microtitre plates (Greiner Labortechnik GmbH, Frickenhausen, Germany) by adding 10ml of a 350nM preparation of Cy3-anti-GST antibody (final assay concentration 70nM) together with 10ml of a 100nM preparation of NF-kB-p65-GST protein (final assay concentration 20nM) in 30ml (total volume) of 10mM HEPES buffer, pH7 containing 0.2mM EDTA, 20mM NaOAc, 0.05% IPEGAL™, 5mM DTT and 1mg/ml BSA and incubating for 60 minutes at room temperature. Following incubation 10ml of a 500nM preparation of Cy5 labeled dsDNA (containing the NF-kB consensus binding sequence) was added (final assay concentration 100nM) together with various concentrations of unlabeled specific or non-specific competitor dsDNA sequence (all working solutions described were dilutions of more concentrated stock solutions into assay buffer, described above). Reactions, in 50ml total assay volume, were incubated for a further 60 minutes before imaging in LEADseeker™ Homogeneous Imaging System for 90 seconds at 540nm excitation and 690nm emission wavelengths. A Well-by-Well correction method was used with 4 standard concentrations of between 0 and 200nM. The standard used for the corrections was the Cy3-Cy5 calibrator.

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Results

Figure 2: NF-KB competition binding assays

Competition for binding of the Cy5 labeled dsDNA to the NF-kB protein in the presence of either unlabeled specific (— ) or non-specific (— ) competitor dsDNA sequence is shown in Figure 2. Values are mean ± SEM (n=3) and are expressed as %B/Bo derived by standard data analysis. For reference maximum signal:noise levels achieved were in the order 1.7 - 1.8:1 (expressed as specific total FRET signal above the background signal observed in the presence of a 500-fold excess of specific inhibitor) and the IC50 value determined for the specific sequence competitor was 65nM.

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References

• MERCURIO, F. and MANNING, A.M. (1999), Multiple signals converging on NF-kB. Curr. Opin. Cell Biol., 11, pp. 226 - 232.
• MANNING, A. (1998), Targeting the NF-kB activation pathway for gene regulating drugs.
  Curr. Opin. Drug Disc. Dev., 1, 147 - 156.