NanoBRET Assay for Epigenetic PPI in Living Cells


The study of protein interactions is fundamental for the understanding of cellular processes such as DNA replication, transcription, translation, cell signal-transduction, etc. The interaction between epigenetic related proteins is an important way of epigenetic regulation critical for cell health and holds promise for development of novel drug therapies since dysfunction modulation of this interaction has been implicated as a critical event in disease development. The identification of small molecule modulators, inhibitors or activators of protein:protein interactions (PPI) remains challenging, largely attributable to the difficulty of establishing robust, high-throughput screening methodologies that can be applied to investigate these interactions in a biologically relevant context of living cells. Bioluminescent resonance energy transfer (BRET) has long been employed to monitor real-time PPIs in live cells, while its general applicability has been hindered by limited sensitivity and narrow dynamic range. A novel BRET platform, NanoBRET, has recently been presented, which is based on the incorporation of a small and extremely bright NanoLuc luciferase into a HaloTag-long-wavelength fluorophore. This highly effective energy donor-acceptor combination enhances BRET performance, and the higher sensitivity enables the method to be applied in high density plates and high-throughput screening.

Creative BioMart presents several NanoBRET assays for the interrogation of epigenetic protein interaction biology in the context of natural chromatin, thus facilitating lead optimization and structure-activity-relationship analysis. Typical PPI like full-length bromodomain (BRD)-containing protein:acetylated histone interaction could be monitored using this method. BRD fragments alone (BRD4, BRD9 and BRPF1) are also included for users that may want to understand the interaction patterns with other histones. Based on NanoBRET technology, potential small molecule inhibitors can be screened and identified according to their cellular efficacy.

Principle and Procedure of NanoBRET Assay at Creative BioMart

NanoBRET assay technology depends on energy transfer from a luminescent donor (NanoLuc luciferase) to a fluorescent acceptor (HaloTag). NanoLuc luciferase and HaloTag protein are fused to the target proteins of interest, and the fusion proteins are expressed at low cellular levels, enabling monitoring and screening of PPI that reflect true cellular physiology. The NanoBRET assay is fully reversible, allowing the investigation of both induction and inhibition of PPI.

First, both NanoLuc and HaloTag fusion vectors are transfected into the appropriate cell lines. Second, the cells are replated into assay plates with fluorescent HaloTag ligand (for experimental samples) and control samples (no fluorescent ligand), respectively. The system is then treated with various concentrations of compounds and the NanoLuc luciferase substrate is added. An instrument capable of measuring dual-filtered luminescence equipped with appropriate filters is applied to detect donor and acceptor signals. Finally, the corrected NanoBRET ratio is calculated, which is a subtraction of NanoBRET ratios of the control samples from the experimental ligand-containing samples.

Procedure of NanoBRET PPI assay

Figure 1. Procedure of NanoBRET PPI assay

NanoBRET assay at Creative BioMart provides a sensitive live-cell method to study protein:protein interactions, which is a highlighted cellular method in boosting your epigenetic drug discovery within a more relevant environment. Our skilled work team is focused on combining the latest, most relevant technologies into cohesive services tailored to your research. Our cutting-edge platform permits us to offer superior services that meet your demands on time and on budget. Please feel free to contact us for more detailed information about our NanoBRET assay.

Reference
1. Machleidt, T.; et al. NanoBRET-a novel BRET platform for the analysis of protein−protein interactions. ACS Chem Biol. 2015, 10: 1797−1804.

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