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PLos ONE 11, e0161930. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions 2016

Vickers, T.A. and Crooke, S.T.

Notes: Proteins fused to NanoLuc® luciferase were used as the energy donor and AlexaFluor conjugated nucleic acids were used as the energy acceptor in BRET assays that measured protein:nucleic acid interactions. The assay was demonstrated with immunopurified proteins, cell homogenates, and in whole cells. NanoLuc® fusion protein construction, expression, and purification were performed using the vectors pFN31K Nluc CMV-neo for amino-terminal clones and pFC32K Nluc CMV-neo for carboxy-terminal clones. BRET assays were performed in white 96-well plates. Alexa-linked anti-sense oligonucleotides at the indicated concentrations were incubated at room temperature for 15 min in 1X binding buffer with 106 RLU/well of immunoprecipitated NLuc fusion protein or whole cell lysate. Following the incubation, NanoGlo® substrate was added at 0.1 μl/well. Readings were performed using a GloMax® Discover System. (4757)

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