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

Muniasamy Neerathilingam, Sai Gandham, Falguni Patel, Mohammad Nasiruddin

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..
Institute of Molecular Medicine (IMM), University of Texas Health Science Center, Houston, USA..

DOI: 10.4236/jasmi.2013.33022                

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)

Conflicts of Interest

The authors declare no conflicts of interest.

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