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Real-Time Protein:Protein Interactions

Interactions between proteins modulate a large number of biological processes—from fleeting interactions during the transduction of a signaling pathway to the assembly of large multiprotein complexes. The extreme brightness and small size of NanoLuc® luciferase make it possible to label proteins with less risk of interfering with natural function, detecting protein interactions in live cells under native conditions.

Detect Protein Interactions with the Complementation-Based NanoBiT® Reporter

Measure Protein Interactions in Real Time

NanoLuc® Binary Technology (NanoBiT) is a two-subunit system based on NanoLuc® luciferase that can be applied to the intracellular detection of protein:protein interactions (PPIs) in live cells. The NanoBiT® PPI System is composed of two small subunits, Large BiT (LgBiT; 18kDa) and Small BiT (SmBiT; 1.3kDA), that are expressed as fusions to target proteins of interest. The LgBiT and SmBiT subunits have been independently optimized for stability and minimal self-association. Interaction of the target proteins facilitates subunit complementation to give a bright, luminescent enzyme.

We provide Nano-Glo® Live Cell Detection Systems for short-term and extended measurement of protein interaction dynamics in real time.

Get Started with NanoBiT®

Adapting NanoBiT to a Biochemical Assay Format

Watch how researchers at the Francis Crick Institute collaborated with Promega to apply NanoBiT technology to a high-throughput, cell-free biochemical format.

Read article
 

Create Stable NanoBiT® Cell Lines

Although transient expression of fusion partners can be sufficient for many protein interaction experiments, stable cell lines are often needed for maximal reproducibility. This poster describes creating stable cell lines with BiBiT-Ready vectors. This approach allows both SmBiT and LgBiT fusion proteins to be transcribed at similar levels from the same locus following random integration of plasmid DNA into the genome.

Order Bi-Directional Vectors

Measure Protein Interactions with the BRET-Based NanoBRET™ Assays

BRET Assay with Low Background and Increased Signal

The study of PPIs with BRET relies on energy transfer from luciferase (BRET donor) attached to one interacting protein partner, to a fluorescent BRET acceptor tagged to another interacting protein partner. The transfer of energy is moderated by the proximity of the two partners. The NanoBRET™ assay is a substantially improved BRET assay that uses NanoLuc® luciferase as the energy donor and HaloTag® protein Labeled with the NanoBRET™ 618 fluorophore as the energy acceptor. The bright, blue-shifted signal from the NanoLuc® donor combined with the far-red-shifted HaloTag® acceptor create a protein interaction assay with optimal spectral overlap, increased signal, and lower background compared to conventional BRET assays.

Design your own assay or choose from a large selection of prebuilt NanoBRET™ PPI assays targeting bromodomain and other epigenetic proteins, transcriptional proteins, signaling protein and kinase assays, and membrane protein assays.

Get Started with NanoBRET™
 

Detecting CRAF-BRAF Interaction Using NanoBRET™ Technology

CRAF and BRAF protein kinases function within Ras-Raf-MAPK pathway signaling and play critical roles in oncogenesis and cancer. This article describes the development of a protein:protein interaction assay for monitoring CRAF-BRAF heterodimerization using NanoBRET™ technology.

Read Article
Protein Protein Interactions using BRET-Based Assay

Additional Resources

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Engineering a Binary Reporter System

Read this paper to learn about the structural optimization of the NanoLuc® Binary Technology (NanoBiT) components.
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Development of a Novel BRET Platform

This paper discusses the development of NanoLuc® and HaloTag® for BRET assays.
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Luminescent Pull-Down for Confirming NanoBRET® PPI

This article provides a method for validating PPI interactions shown with NanoBRET® using the pull-down functionality of the HaloTag® protein.
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