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Investigation of Protein:Protein Interactions (PPI) using a novel Bioluminescence Resonance Energy Transfer (BRET) Proximity-based, High-Throughput Screening Assay下載
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February 03, 2016
Authors: Ellaine Abueg, P. Brescia, P. Banks, BioTek Instruments, Inc. Winooski, VT USA; A. Landreman, Promega Corporation, Madison, WI USA
Proteins have been found to play a variety of critical roles in living cells. To better understand how individual proteins function, it is important to be able to elucidate the dynamic interactions occurring within a cellular context. While several methods exist to investigate protein:protein interactions (PPI) in living cells, monitoring proves to be difficult. Here we demonstrate the use of a new PPI system similar in configuration and approach to bioluminescence resonance energy transfer (BRET). The system is a proximity-based assay that relies on the measure of energy transfer from a bioluminescent donor protein to a fluorescently tagged acceptor protein (Figure 1). The optimized blue-shifted donor and redshifted acceptor pair helps to minimize both assay background due to the biological nature of the sample and from spectral overlap (Figure 2). Preliminary data includes analysis of a donoracceptor fusion protein for assay and instrumentation optimization as well as investigation of the donor-MDM2 and acceptor-p53 fusion pair in a cell-based system. The MDM2 and p53 fusion protein pair was transiently transformed into HEK293 cells for analysis including Z’- factor and pharmacological studies.
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