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. The NanoLuc® technology has been further adapted to enable sensitive bioluminescent detection of protein interactions in biochemical assay formats.

Measure Live-Cell Protein Interactions in Real Time

Detect Protein Interactions with the Complementation-Based NanoBiT® Reporter

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.

Real-time monitoring of EGFR(1-673) dimerization following stimulation with increasing concentrations of EGF. The NanoBiT® EGFR(1-673) fusion constructs were stably expressed in U20S cells using the BiBiT bi-directional expression system.

Design Your Own: Use our NanoBiT® PPI Starter Systems to develop your own assay for studying target proteins of interest.

Design Your Own NanoBiT® Assay

Research Highlights

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

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Read how researchers have applied NanoBiT® technology to develop high-throughput methods for screening and continuous monitoring of endogenous protein interactions in real time.

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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.

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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.

Application Example

Endogenously expressed proteins tagged with HiBiT using CRISPR/CAS9 gene editing can also be used as the BRET energy donor when LgBiT is also expressed in the cell. The HaloTag® fusion can be introduced transiently allowing for interactions kinetics to be assessed under native expression conditions.

Here is an example of an endogenously tagged protein, HiBiT-BRD4, with varying kinetic increases in ubiquitination levels that depend on specific PROTAC treatment.

NanoBRET® PPI Assay Options

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. Additional tools are available to develop NanoBRET® assays for measuring ternary complex formation and target protein ubiquitination.

Design Your Own: Use our NanoBRET® PPI Starter systems to develop your own assay for studying target proteins of interest.

Design Your Own NanoBRET® Assay

Use Predesigned Assays: A variety of pre-designed, optimized assays for popular protein interactions such RAS signaling, β-arrestin recruitment and epigenetic signaling are available in NanoBRET® and NanoBiT® formats.

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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.

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Protein Protein Interactions using BRET-Based Assay

Measure Protein Interactions with an Immunoassay Format

lumit-anti-tag-protein-protein-interaction-fig2

Assay principle. The Lumit® Anti-Tag PPI immunoassay is a homogeneous (no-wash) assay system to measure PPIs on purified proteins, combining immunodetection with NanoBiT® technology. In the Lumit® PPI Immunoassay, antibodies against commonly used tags 6His, GST, FLAG® and human-Fc are labeled with LgBiT or SmBiT. When two proteins with different tags interact, the interaction can be detected using the corresponding SmBiT- and LgBiT-conjugated antibodies, bringing the two NanoBiT® subunits together to reconstitute the active bioluminescent enzyme.

Detection of GTP-Dependent KRAS-cRAF Interaction

Assay principle. KRAS interacts with downstream effector cRAF when bound to GTP or non-hydrolyzable analog GppNHp.

Lumit® PPI assays are sensitive enough to detect small changes in GTP-bound state.

Interested in using Lumit® immunoassays to study protein interactions?

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Protein:Protein Interactions

Measure Live-Cell Protein Interactions in Real Time