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Determination of Interaction between NFκB p50 and <i>β</i>-IFN-<i>κ</i>B Binding Oligo Using AlphaLISA in HTP Fashion
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.
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
- 1 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.
- 2 Institute of Molecular Medicine (IMM), University of Texas Health Science Center, Houston, USA.
- 3 Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India
- 4 Centre for Cellular Molecular Platforms (C-CAMP), NCBS-TIFR, GKVK Campus, Bangalore, India
Journal of Analytical Sciences, Methods and Instrumentation·Volume 03 (2013)·Pages 173–178·Published 23 August 2013·DOI10.4236/jasmi.2013.33022
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Abstract
NF- κ B 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 NF κ B intraction with β -IFN- κ B binding oligo. Each of these techniques ha s varying utility with distinct strengths and weaknesses. We describe a method AlphaLISA to identify NF κ B p50 protein and β -IFN- κ B 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.
KeywordsDNA-Protein InteractionBinding ConstantEquilibrium ConstantAlphaLISAHigh Throughput (HTP)
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