A Homogeneous TR-FRET NF-κB Protein:DNA Binding Assay Introduction Figure 1. NF-κB binding assay schematic Excitation at 340nm Eu3+ 340/35nm excitation and 670/11nm emission filter sets. Lag time was set at 50µS, integration time was 400µS with 30 flashes/well. DMSO is a solvent commonly used in assay buffers and preparations. Exposing the sample wells in an assay to varying amounts of the solvent was performed to evaluate of the tolerance of the assay to DMSO. DMSO was added to the sample wells at a maximal concentration of 10% of the well volume. TR-FRET P65 GST 40000 0.0 Lowest limit of detection (sensitivity) of NF-κB specific dsDNA in the assay was 5.2fmol/well (52pM) as determined by titration with 10nM recombinant protein (figure 2). At maximal acceptor concentration (40nM), signal:noise for the assay was 9.6:1. 5.0 6.0 7.0 8.0 9.0 10.0 Z’ factor analysis was performed as described by Zhang et al4. Results of the analysis are shown in figure 6. Assays with a Z’ factor between 0.5 and 1 are considered to be robust and reliable. The NF-κB protein:DNA binding assay that we have developed has a Z’ factor of 0.87. Figure 5. IκBα protein inhibition. 1 0 0 8 0 30000 7 0 3 L o g 20000 10000 0 0 1 2 3 1 0 4 Iκ B 5 c o n c e n t r a t io n (p M ) IκBα protein was titrated with a constant 10nM p65-GST recombinant protein at a maximum concentration of 100nM. IC50 value determined from the assay was 15nM. Data was plotted as triplicates, mean ± SEM. S e n si t i v i t y = 5 . 2 f m o l / w e l l 4 Figure 6. Z’ Analysis. p m o l/w e ll N F κ B s p e c if ic dsDNA 60000 Specific biotinylated dsDNA was titrated from 40nM with a constant 10nM p65GST and europium chelate labelled anti-GST antibody. Cy5 labelled streptavidin acceptor was added to the biotinylated dsDNA at a 1:2 (w/w) concentration. Data plotted as quadruplicates, mean ± SEM. Figure 3. NF-κB binding competition. TR-FRET RFU TR-FRET RFU 40000 Oligonucleotide sequences were prepared ‘in house’ using standard phosphoramidite chemistry. The 19 base-pair stretch of double-stranded DNA’s yielded are shown in Table 1. 40000 20000 100 0 0 80 20 40 60 80 100 120 140 %B/B0 R e p l ic a t e 60 10nM of p65-GST recombinant protein was incubated with 10nM of Eu anti-GST antibody and 10nM Cy5 streptavidin in the presence () or absence (À) of NF-κB specific dsDNA. Z’ factor analysis was evaluated over 140 sample wells. Z’ = 0.87. 40 20 0 Evaluation of the assay to DMSO tolerance showed that no significant effect was evident even at the highest DMSO content of 10% of the volume in the well (figure 4). CONCLUSION We have demonstrated the ability to measure • NF-κB specific dsDNA binding to a p65-GST recombinant protein by TR-FRET using generic reagents and measuring on FARCyte. The assay is both robust and sensitive using • generic TR-FRET reagents. The results highlight the potential of the • FARCyte Fluorescence Plate Reader for measurement of sensitive TR-FRET assays. Inhibition of protein:dsDNA binding was evaluated using IκBα protein. IκBα is a NFκB regulatory protein found in the cell cytoplasm which inhibits its DNA binding activity. Effect of inhibition by IκBα is shown in figure 5 and demonstrates that increasing concentrations of the inhibitory protein reduces the amount of signal in the assay as less binding occurs. IC50 values for the inhibition of NFκB specific dsDNA binding to the p65 recombinant protein by IκBα in the assay was 15nM. References 1. Bours, V., et al., Biochemical Pharm., 60, 1085-1090, (2000). 2. Baldwin, A. S., J. Clin. Invest. 107, 1, 3-6, (2001). 3. Jones, S. G., et al., J. Fluoresc. 11,1,13-21, (2001) 4. Zhang, J-H., et al., J Biomol Screening, 2, 6773 (1999). 2 NF-κB p65-GST recombinant protein (10nM) was incubated with Eu anti-GST fusion protein (10nM) in the dark with agitation. The reaction mixture was incubated for 1 hour at room temperature (20-25oC) in 10mM HEPES, 20mM Sodium Acetate, 0.2mM EDTA buffer, pH 7.0 containing 5mM DTT, 1mg/ml BSA and 0.05% NP40. Sensitivity was evaluated using a 2n titration of 40nM of biotinylated NF-κB specific dsDNA. For competition, inhibition, DMSO tolerance and Z’ analysis assays, 20nM biotinylated NF-κB specific dsDNA was added to the reaction mix and incubated at room temperature for a further 1 hour in the dark with agitation. Finally, Cy5 streptavidin (10nM) was added to each reaction well and further incubated for 15 minutes. Reactions were performed in a total volume of 100µl using Corning black 384-well NBS plates. TR-FRET was measured on the FARCyte fluorescence plate reader using 4.0 Evaluation of the effect of DMSO solvent on the assay signal. Data is plotted as quadruplicates ± SEM. Cy5 labelled streptavidin Oligo sequences were prepared using standard phosphoramidite chemistry and purified by C18 reverse phase HPLC. Double-stranded DNA was prepared by incubating equimolar amounts of the NF-κB-specific (HIV-L) coding (biotinylated or non-biotinylated) and unmodified non-coding strands in o a 75 C water bath for 3-5 minutes before allowing to cool to ambient temperature. 3.0 %B/B0 Biotin dsDNA Unlabelled NF-κB-specific competitor dsDNA 5’ GATCTAGGGACTTTCCGCG 3’ 3’ ATCCCTGAAAGGCGCCTAG 5’ Unlabelled non-specific competitor dsDNA 5’ GATCTATTGACTTAAGTG 3’ 3’ ATAACTGAATTCACCTAG 5’ 2.0 9 0 50000 Table 1. NF-κB assay dsDNA Biotinylated dsDNA (NF-κB-specific) 5’ Biotin-GATCTAGGGACTTTCCGCG 3’ 3’ AT CCCTGAAAGGCGCCTAG 5’ 1.0 % DM SO Figure 2. NF-κB assay sensitivity of binding. Method 20000 0 Emission at 665nm Assay is configured using a europium chelate labelled anti-GST antibody specific for a p65-GST recombinant protein that interacts with a biotinylated NFκB-specific dsDNA bound to Cy5 labelled streptavidin 30000 10000 Results Competition of binding to the p65-GST protein (figure 3) demonstrated that signal is significantly reduced with the addition of the specific dsDNA competitor containing the NF-κB p65 consensus binding sequence, whereas the non-specific dsDNA reduced signal only marginally when added at considerable excess, demonstrating binding specificity. TMT(Eu) Labelled Anti-GST Figure 4. Effect of DMSO on the assay. TR-FRET RFU Nuclear Factor-κB (NF-κB) is a transcription factor that is considered to be of physiological importance because of its key role as a regulatory molecule involved in immune response, inflammation, cancer and apoptosis1,2. We have developed a time resolved-fluorescence resonance energy transfer (TR-FRET) assay to evaluate the binding interaction between the p65 subunit of NF-κB and a dsDNA NFκB-specific (HIV-L) consensus sequence. The development of a TR-FRET NF-κB binding assay has been reported previously3 using direct labelled p65 specific dsDNA. We have now further developed the assay to incorporate generic europium [Eu(TMT)] donor and Cy5 acceptor reagents (figure 1). 3 Lo g 10 4 5 6 7 d s D N A c o n c e n tr a t io n (p M ) Competition of the biotinylated dsDNA for binding to the p65-GST recombinant protein was demonstrated using unlabelled NF-κB specific dsDNA sequence () and an unlabelled non-specific dsDNA sequence (À). dsDNA was titrated from a maximum concentration of 1µM. Data plotted as triplicates, mean ± SEM. Copyright ©2009, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. 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