Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion
Affiliation(s)
Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India&Institute of Molecular Medicine (IMM), University of Texas Health Science Center, Houston, USA.. Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India.
Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India&Institute of Molecular Medicine (IMM), University of Texas Health Science Center, Houston, USA.. Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India.
ABSTRACT
NF-κB
plays a crucial role in regulating various biological processes including
innate and adaptive immunity, inflammation, stress responses, B-cell
development, and lymphoid organogenesis. Currently, several assays like
electrophoretic mobility shift assay (EMSA), enzyme-linked immunosorbent assay
(ELISA), fluorescence resonance energy transfer (FRET) and time-resolved
fluorescence resonance energy transfer (TR-FRET) are widely used for studying
the NFκB intraction with β-IFN-κB binding oligo.
Each of these techniques has varying utility with distinct strengths and weaknesses. We describe a method AlphaLISA to identify
NFκB p50 protein and β-IFN-κB binding oligo sequence and interaction is efficient at a given
concentration (10 nM) in the EMSA and Biacore’s SPR assays. The method has many
advantages such as use of small volume, high throughput (HTP), convenience of
sample preparation and data analysis.
KEYWORDS
DNA-Protein Interaction; Binding Constant; Equilibrium Constant; AlphaLISA; High Throughput (HTP)
DNA-Protein Interaction; Binding Constant; Equilibrium Constant; AlphaLISA; High Throughput (HTP)
Cite this paper
M. Neerathilingam, S. Gandham, F. Patel and M. Nasiruddin, "Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion," Journal of Analytical Sciences, Methods and Instrumentation, Vol. 3 No. 3, 2013, pp. 173-178. doi: 10.4236/jasmi.2013.33022.
M. Neerathilingam, S. Gandham, F. Patel and M. Nasiruddin, "Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion," Journal of Analytical Sciences, Methods and Instrumentation, Vol. 3 No. 3, 2013, pp. 173-178. doi: 10.4236/jasmi.2013.33022.
References
[1] S. Blouin, T. D. Craggs, D. A. Lafontaine and J. C. Penedo, “Functional Studies of DNA-Protein Interactions Using FRET Techniques,” Methods in Molecular Biology, Vol. 543, 2009, pp. 475-502. http://dx.doi.org/10.1007/978-1-60327-015-1_28 [2] D. J. King, S. E. Bassett, X. Li, S. A. Fennewald, N. K. Herzog, B. A. Luxon, R. Shope and D. G. Gorenstein, “Combinatorial Selection and Binding of Phosphorothioate Aptamers Targeting Human NF-Kappa B RelA (p65) and p50,” Biochemistry, Vol. 41, No. 30, 2002, pp. 9696-9706. http://dx.doi.org/10.1021/bi020220k [3] G. Courtois and T. D. Gilmore, “Mutations in the NF-κB Signaling Pathway: Implications for Human Disease,” Oncogene, Vol. 25, 2006, pp. 6831-6843. [4] M. Karin, “Nuclear Factor-κB in Cancer Development and Progression,” Nature, Vol. 441, 2006, pp. 431-436. http://dx.doi.org/10.1038/nature04870 [5] R. G. Baker, M. S. Hayden and S. Ghosh, “NF-κB, Inflammation and Metabolic Disease,” Cell Metabolism, Vol. 13, No. 1, 2011, pp. 11-22. http://dx.doi.org/10.1016/j.cmet.2010.12.008 [6] A. Kumar, Y. Takada, A. M. Boriek and B. Aggarwal, “Nuclear Factor-κB: Its Role in Health and Disease,” Journal of Molecular Medicine, Vol. 82, No. 7, 2004, pp. 434-448. http://dx.doi.org/10.1007/s00109-004-0555-y [7] N. D. Perkins, “Integrating Cell-Signalling Pathways with NF-κB and IKK Function,” Nature Reviews Molecular Cell Biology, Vol. 8, No. 1, 2007, pp. 49-62. http://dx.doi.org/10.1038/nrm2083 [8] F. Arenzana-Seisdedos, P. Turpin, M. Rodriguez, et al., “Nuclear Localization of IkBa Promotes Active Transport of NF-kB from the Nucleus to the Cytoplasm,” Journal of Cell Science, Vol. 110, 1997, pp. 369-378. [9] Gel Shift Assays—EMSA, Protein Methods Library, Thermo Scientific. [10] C. W. Muller and S. C. Harrison, “The Structure of the NF-Kappa B p50: DNA-Complex: A Starting Point for Analyzing the Rel Family,” FEBS Letter, Vol. 369, No. 1, 1995, pp. 113-117. http://dx.doi.org/10.1016/0014-5793(95)00541-G [11] TR-FRET Basics. CISBIO Bioassays. [12] J. Paulo, “BIACORE T100: An Overview of Underlying Concepts, Basic Operation, Experimental Techniques and Potential Applications,” JP Biacore Presentation, 071106. [13] L. Beaudet, R. Rodriguez-Suarez, M. H. Venne, M. Caron, J. Bedard, V. Brechler, S. Parent and M. Bielefeld-Sevigny, “AlphaLISA Immunoassays: The No-Wash Alternative to ELISAs for Research and Drug Discovery,” Nature Methods, Vol. 5, 2008. [14] PerkinElmer’s AlphaLISA. Bead Selection and Bead Interference—Assay Support Knowledge Base. [15] R. Eglen and S. Howland, “AlphaLISA in Biomarker Detection for Drug Discovery,” Technology & Services Section, PerkinElmer, Inc. [16] D. E. Volk, X. Yang, S. M. Fennewald, D. J. King, S. Bassett, S. Venkitachalam, N. Herzog, B. A. Luxon and D. G. Gorenstein, “Solution Structure and Design of Dithiophosphate Backbone Aptamers Targeting Transcription Factor NF-Kappa B,” Bioorganic Chemistry, Vol. 30, No. 6, 2002, pp. 396-419. http://dx.doi.org/10.1016/S0045-2068(02)00510-2 [17] T. Fujita, G. P. Nolan, S. Ghosh and D. Baltimore, “Independent Modes of Transcriptional Activation by the p50 and p65 Subunits of NF-Kappa B,” Genes & Development, Vol. 6, 1992, pp. 775-787. http://dx.doi.org/10.1101/gad.6.5.775 [18] D. Thanos and T. Maniatis, “The High Mobility Group Protein HMG I (Y) Is Required for NF-Kappa B-Dependent Virus Induction of the Human IFN-Beta Gene,” Cell, Vol. 71, No. 5, 1992, pp. 777-789. http://dx.doi.org/10.1016/0092-8674(92)90554-P [19] PerkinElmer’s AlphaLISA. Determining KD with an Alpha Assay. [20] PerkinElmer’s AlphaLISA. The Hook Effect—Assay Support Knowledge Base.
[1] S. Blouin, T. D. Craggs, D. A. Lafontaine and J. C. Penedo, “Functional Studies of DNA-Protein Interactions Using FRET Techniques,” Methods in Molecular Biology, Vol. 543, 2009, pp. 475-502. http://dx.doi.org/10.1007/978-1-60327-015-1_28 [2] D. J. King, S. E. Bassett, X. Li, S. A. Fennewald, N. K. Herzog, B. A. Luxon, R. Shope and D. G. Gorenstein, “Combinatorial Selection and Binding of Phosphorothioate Aptamers Targeting Human NF-Kappa B RelA (p65) and p50,” Biochemistry, Vol. 41, No. 30, 2002, pp. 9696-9706. http://dx.doi.org/10.1021/bi020220k [3] G. Courtois and T. D. Gilmore, “Mutations in the NF-κB Signaling Pathway: Implications for Human Disease,” Oncogene, Vol. 25, 2006, pp. 6831-6843. [4] M. Karin, “Nuclear Factor-κB in Cancer Development and Progression,” Nature, Vol. 441, 2006, pp. 431-436. http://dx.doi.org/10.1038/nature04870 [5] R. G. Baker, M. S. Hayden and S. Ghosh, “NF-κB, Inflammation and Metabolic Disease,” Cell Metabolism, Vol. 13, No. 1, 2011, pp. 11-22. http://dx.doi.org/10.1016/j.cmet.2010.12.008 [6] A. Kumar, Y. Takada, A. M. Boriek and B. Aggarwal, “Nuclear Factor-κB: Its Role in Health and Disease,” Journal of Molecular Medicine, Vol. 82, No. 7, 2004, pp. 434-448. http://dx.doi.org/10.1007/s00109-004-0555-y [7] N. D. Perkins, “Integrating Cell-Signalling Pathways with NF-κB and IKK Function,” Nature Reviews Molecular Cell Biology, Vol. 8, No. 1, 2007, pp. 49-62. http://dx.doi.org/10.1038/nrm2083 [8] F. Arenzana-Seisdedos, P. Turpin, M. Rodriguez, et al., “Nuclear Localization of IkBa Promotes Active Transport of NF-kB from the Nucleus to the Cytoplasm,” Journal of Cell Science, Vol. 110, 1997, pp. 369-378. [9] Gel Shift Assays—EMSA, Protein Methods Library, Thermo Scientific. [10] C. W. Muller and S. C. Harrison, “The Structure of the NF-Kappa B p50: DNA-Complex: A Starting Point for Analyzing the Rel Family,” FEBS Letter, Vol. 369, No. 1, 1995, pp. 113-117. http://dx.doi.org/10.1016/0014-5793(95)00541-G [11] TR-FRET Basics. CISBIO Bioassays. [12] J. Paulo, “BIACORE T100: An Overview of Underlying Concepts, Basic Operation, Experimental Techniques and Potential Applications,” JP Biacore Presentation, 071106. [13] L. Beaudet, R. Rodriguez-Suarez, M. H. Venne, M. Caron, J. Bedard, V. Brechler, S. Parent and M. Bielefeld-Sevigny, “AlphaLISA Immunoassays: The No-Wash Alternative to ELISAs for Research and Drug Discovery,” Nature Methods, Vol. 5, 2008. [14] PerkinElmer’s AlphaLISA. Bead Selection and Bead Interference—Assay Support Knowledge Base. [15] R. Eglen and S. Howland, “AlphaLISA in Biomarker Detection for Drug Discovery,” Technology & Services Section, PerkinElmer, Inc. [16] D. E. Volk, X. Yang, S. M. Fennewald, D. J. King, S. Bassett, S. Venkitachalam, N. Herzog, B. A. Luxon and D. G. Gorenstein, “Solution Structure and Design of Dithiophosphate Backbone Aptamers Targeting Transcription Factor NF-Kappa B,” Bioorganic Chemistry, Vol. 30, No. 6, 2002, pp. 396-419. http://dx.doi.org/10.1016/S0045-2068(02)00510-2 [17] T. Fujita, G. P. Nolan, S. Ghosh and D. Baltimore, “Independent Modes of Transcriptional Activation by the p50 and p65 Subunits of NF-Kappa B,” Genes & Development, Vol. 6, 1992, pp. 775-787. http://dx.doi.org/10.1101/gad.6.5.775 [18] D. Thanos and T. Maniatis, “The High Mobility Group Protein HMG I (Y) Is Required for NF-Kappa B-Dependent Virus Induction of the Human IFN-Beta Gene,” Cell, Vol. 71, No. 5, 1992, pp. 777-789. http://dx.doi.org/10.1016/0092-8674(92)90554-P [19] PerkinElmer’s AlphaLISA. Determining KD with an Alpha Assay. [20] PerkinElmer’s AlphaLISA. The Hook Effect—Assay Support Knowledge Base.