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.