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Studying the interplay between proteins in living cells allows for the development of compounds designed to inhibit protein-protein interactions (PPI) to modulate biological processes in normal and disease tissue. We have successfully used NanoBRET™ System (Promega) to detect protein- protein interactions in living cells. NanoBRET™ is a proximity-based assay that can detect protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. The use of full-length proteins expressed at low levels enables PPI monitoring and screening studies that reflect true cellular physiology. NanoBRET™ is a reversible assay which makes it possible to study both induction and inhibition of protein interactions.

Examples:

Using this system, we have screened for inhibitors of the Schnurri-3 (Shn-3) ERK-2 PPI in HEK-293 cells.

Competition of MEK protein with Shn-3 for binding to ERK-2

Competition of an ERK-2 inhibitor binding to ERK-2 and preventing the binding of Shn-3

Frequency Distribution of Compound Inhibition of the Shn-3–ERK-2 Interaction

Z’ Factors for the 56 x 384 well plates screened in the NanoBRET Shn-3–ERK-2 assay

Retesting of compounds from primary assay

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