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Gel Shift Assays—EMSA, Protein Methods Library, Thermo Scientific.

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• TITLE: Determination of Interaction between NFκB p50 and β-IFN-κB Binding Oligo Using AlphaLISA in HTP Fashion

  AUTHORS: Muniasamy Neerathilingam, Sai Gandham, Falguni Patel, Mohammad Nasiruddin

  KEYWORDS: DNA-Protein Interaction; Binding Constant; Equilibrium Constant; AlphaLISA; High Throughput (HTP)

  JOURNAL NAME: Journal of Analytical Sciences, Methods and Instrumentation, Vol.3 No.3, September 26, 2013

  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.