Traditional techniques for studying protein:protein interactions (e.g., affinity capture or mass spectrometry) are limited in that they present a static picture of what is a dynamic process. To gain a more accurate and complete picture of interactions, they need to be studied n the context of the cellular environment within which they occur. In the webinar: Monitoring Protein:Protein Interactions in Living Cells Using NanoBRET™ Technology, Dr. Danette Daniels presented an improved Bioluminescence Resonance Energy Transfer (BRET) Technology that was developed to specifically study dynamic interactions in living cells. The NanoBRET™ Technology combines the small, extremely bright NanoLuc® Luciferase as the donor with a fluorescently labeled HaloTag® protein fusion tag as the acceptor to form a BRET assay. This assay offers an increased signal and decreased spectral overlap compared to other BRET technologies. Among other things, these features provide a larger signal range and the brightness of the NanoLuc® Luciferase enables protein:protein interaction analysis even with the donor protein expressed at low levels.
In her webinar, Dr. Daniels demonstrated the advantages of the NanoBRET™ Technology using bromodomains. Bromodomains are protein interaction modules that specifically bind to acetylated lysine residues such as those on the N-terminal tails of histones. This recognition is a key event in epigenetic marks and is associated with protein-histone association and chromatin remodeling. The bromodomain family is interesting because it is implicated in numerous diseases and has been identified as a promising target for compound testing. Current assays use a purified domain and histone fragments to evaluate compound performance. Creating in vitro assays as proven difficult in part because of the complexity of the chromatin.
Using a NanoBRET™ Assay, Dr. Daniels was able to monitor histone interactions with chromatin and observe chromatin incorporation of histone HaloTag® fusions in vitro. She was also able to detect the specific interaction of the bromodomain BRD4 with histones H3.3 and H4. She also showed a panel measuring the interaction of full-length bromodomains with the H3.3. Using a bromodomain and extra-terminal domain (BET) inhibitor, the NanoBRET™ Assay was also able to measure the potency of the inhibitor with different bromodomain and histone combinations.
Dr. Daniels’ webinar highlighted several additional applications for the NanoBRET™ Technology, including studying compound binding to target proteins, ranking the potency of compounds, determining the specificity of binding, creating competition profiles for compounds as well as imaging and protein localization experiments.
About the Webinar Series
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