NanoBiT® Complementation Assay for Protein Interactions

NanoBiT - Go Further Than You Ever Have Before

NanoLuc® Binary Technology (NanoBiT) is a structural complementation reporter designed for protein:protein interaction (PPI) studies. NanoBiT has two small subunits optimized for stability, low self-affinity and bright luminescence. If two proteins of interest are tagged with these subunits and then interact, the subunits come together to form an active enzyme. The proportional signal-to-activity and plate-based format make NanoBiT ideal for PPI studies, including high-throughput screening.

Design PPI assays that more accurately reflect natural cellular biology. Small subunit size (LgBiT, 18kDa and SmBiT, 11 amino acids) minimizes interference with normal protein function, and the bright signal accommodates low, native expression levels. NanoBiT® subunit interactions are reversible and can detect rapidly associating and dissociating proteins. Use NanoBiT in live cells for accurate, real-time monitoring of protein dynamics within a natural cellular context.

Read more about NanoBiT® technology in Dixon et al., 2015:
NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells

Order a Custom AssayOrder Custom NanoBiT® Vectors

In addition to NanoBiT® PPI Starter Systems available from our catalog you can special order NanoBiT® Vectors with different promoter options from Promega Custom Assay Services.

Order Custom NanoBiT® Vectors

Exciting possibilities for exploring protein interactions in live cells

The optimized NanoBiT® reporters open up a world of options for exploring natural biology. See how NanoBiT® technology is inspiring scientists to take their research a little BiT further in the following videos.

Watch Protein Interactions in Real Time Inside Cells

HeLa cells were transfected with FKBP-SmBiT and FRB-LgBiT fusion constructs. Following overnight incubation, substrate was added and samples imaged on an LV200 microscope. After baseline read for approximately 30 seconds, 500nM rapamycin was added to induce FKBP:FRB interaction and images were recorded (imaging cycle = 0.5 seconds).

 

Monitor Induction of Protein Interactions

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HEK293 cells were transfected with LgBiT and SmBiT fusions to the androgen receptor (AR), followed by overnight incubation. Panel A, For kinetic measurement of AR dimerization, luminescence was measured for 30 minutes at 37°C. Wells were then injected with either vehicle or 100nM final concentration of R1881 to stimulate dimerization and luminescence measured for 60 minutes. Panel B. For endpoint analysis of AR dimerization, wells were injected with varying concentrations of R1881 and luminescence measured 30 minutes after compound addition.

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HEK293 cells were transfected with LgBiT and SmBiT fusions to the androgen receptor (AR), followed by overnight incubation. Panel A, For kinetic measurement of AR dimerization, luminescence was measured for 30 minutes at 37°C. Wells were then injected with either vehicle or 100nM final concentration of R1881 to stimulate dimerization and luminescence measured for 60 minutes. Panel B. For endpoint analysis of AR dimerization, wells were injected with varying concentrations of R1881 and luminescence measured 30 minutes after compound addition.

Monitor Inhibition of Protein Interactions

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HEK293T cells were transfected with LgBiT-p53, SmBiT-MDM2 fusion constructs. Following overnight incubation cells were treated with inhibitor Nutlin-3 at the indicated concentrations at room temperature for 4 hours followed by to reagent addition and luminescence measurement.

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HEK293T cells were transfected with LgBiT-p53, SmBiT-MDM2 fusion constructs. Following overnight incubation cells were treated with inhibitor Nutlin-3 at the indicated concentrations at room temperature for 4 hours followed by to reagent addition and luminescence measurement.

Understanding the NanoBiT® PPI System

NanoBiT is a structural reporter that uses two small complementary subunits as fusion partners with proteins of interest. When those proteins interact, the two subunits of NanoBiT are brought together to form an active enzyme.

Exploring PPI with SmBiT: LgBiT Fusions

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Improved Brightness and Accuracy Over Split Firefly Luciferase.

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The FKBP:FRB model system was used with HEK293 cells transfected with either NanoBiT or firefly luciferase fusion constructs. After overnight incubation cells were treated with rapamycin to stimulate FKBP:FRB interaction. Panel A. Cells were treated with rapamycin as indicated and incubated at room temperature (21°C) two hours followed by reagent addition and luminescence measurement. Panel B. After reagent addition, cells were incubated at 37°C and luminescence measured for approximately 30 minutes. Cells were then treated with 30nM rapamycin and luminescence monitored for an additional 40 minutes.

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The FKBP:FRB model system was used with HEK293 cells transfected with either NanoBiT or firefly luciferase fusion constructs. After overnight incubation cells were treated with rapamycin to stimulate FKBP:FRB interaction. Panel A. Cells were treated with rapamycin as indicated and incubated at room temperature (21°C) two hours followed by reagent addition and luminescence measurement. Panel B. After reagent addition, cells were incubated at 37°C and luminescence measured for approximately 30 minutes. Cells were then treated with 30nM rapamycin and luminescence monitored for an additional 40 minutes.

A Dynamic, Reversible System

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HEK293 cells were transfected with SmBiT-PRKACA and LgBiT-PRKAR2A control vectors. After overnight incubation cells were treated to modulate cAMP levels: Isoproterenol (ADRB2 agonist); Propranolol (ADRB2 antagonist); and Forskolin (activator of adenylate cyclase). The NanoBiT™ PRKACA:PRKAR2 interaction was readily reversible showing rapid dissociation in response to increased cAMP and rapid association in response to decreased cAMP. Changes in intracellular cAMP were also monitored independently using the GloSensor™ cAMP Assay, which demonstrates the expected inverse correlation to the PRKACA:PRKAR2 interaction.

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HEK293 cells were transfected with SmBiT-PRKACA and LgBiT-PRKAR2A control vectors. After overnight incubation cells were treated to modulate cAMP levels: Isoproterenol (ADRB2 agonist); Propranolol (ADRB2 antagonist); and Forskolin (activator of adenylate cyclase). The NanoBiT™ PRKACA:PRKAR2 interaction was readily reversible showing rapid dissociation in response to increased cAMP and rapid association in response to decreased cAMP. Changes in intracellular cAMP were also monitored independently using the GloSensor™ cAMP Assay, which demonstrates the expected inverse correlation to the PRKACA:PRKAR2 interaction.

Brightness Enables Single Copy Integration.

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HEK293 cells containing stable integration of BRAF-LgBiT and CRAF-SmBiT fusions expressed by a bidirectional CMV promoter were plated into 96-well plates. Cells were starved two hours, then treated with GDC0879 for 4 hours as indicated to induce dimerization, and luminescence was measured.

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HEK293 cells containing stable integration of BRAF-LgBiT and CRAF-SmBiT fusions expressed by a bidirectional CMV promoter were plated into 96-well plates. Cells were starved two hours, then treated with GDC0879 for 4 hours as indicated to induce dimerization, and luminescence was measured.