NanoBiT® PPI Starter Systems
Sensitive Detection of Protein Interactions in Live Cells
- Detects interactions at low expression levels and allows real-time kinetic analysis in live cells
- Reversible protein complementation reporter to analyze protein association and disassociation
- Improved sensitivity and accuracy over split firefly luciferase
Catalog Number:
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Catalog Number: N2014
Catalog Number: N2015
Catalog Number: N2016
Protein Interaction Assays that Reflect Natural Cellular Biology
NanoLuc® Binary Technology (NanoBiT) is a structural complementation reporter system composed of a Large BiT (LgBiT; 18kDa) subunit and a small complementary peptide. For the study of protein:protein interactions, the complementary peptide is Small BiT (SmBiT; 11 amino acid peptide), which has been optimized to have low affinity for LgBiT. The LgBiT and SmBiT subunits are expressed as fusions to target proteins of interest and expressed in cells. When the two proteins interact, the subunits come together to form an active enzyme and generate a bright luminescent signal in the presence of substrate.
The small size of the NanoBiT® complementation partners minimizes interference with normal protein function, and the bright signal accommodates low, native expression levels. The low self-affinity of the NanoBiT® subunits combined with live-cell detection offers kinetic, real-time analysis of protein interaction dynamics.
Study Drug Potency and Binding Kinetics
The proportional signal-to-activity and plate-based format make NanoBiT ideal for PPI studies, including high-throughput screening.
"NanoBiT can reveal drug potency for induced protein interactions and the binding kinetics underlying drug action"
You can read the technical details behind NanoBiT technology and review example data from protein interaction assays in the paper: NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells, Dixon et al., (2016) ACS Chem. Biol., 11, 400–408.
Monitor Induction and Inhibition of Protein Interactions with a Dynamic, Reversible Assay
In contrast to many split systems, the LgBiT:SmBiT interaction is reversible, allowing the detection of rapidly dissociating proteins. PPI dynamics can be followed in real time in living cells using the Nano-Glo® Live Cell Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate. Advantages over split systems include better sensitivity, reversibility, the ability to create fusions to peptides or small, structurally stable protein domains, and the capability for real-time measurement of protein interactions.
The poster Monitoring Intracellular Protein Interactions using NanoBiT Binary Technology provides more details, including data illustrating these capabilities.
Monitor Protein Dynamics in Real Time
In this experiment, 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).
Example NanoBiT Applications
NanoBiT® System Vectors
Using the NanoBiT® MCS Starter System, you can generate N- and C-terminal LgBiT and SmBiT fusions to proteins of interest using traditional cloning with a multiple cloning site (MCS). Expression is driven by HSV-TK promoter, providing constitutive, low-level expression in mammalian cells.
The NanoBiT® Flexi® Starter System, allows you to generate N- and C-terminal LgBiT and SmBiT fusions using the Flexi® Vector Cloning System, a directional cloning method based on the rare-cutting restriction enzymes, SgfI and PmeI, that provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between Flexi® Vectors without the need to resequence. The Find My Gene™ service contains many ORF clones already in Flexi® format for simple creation of fusions.
The NanoBiT® PPI Control Pair (FKBP:FRB; Cat.# N2016) is available separately as an inducible positive control.
Choose Starter Systems, Prebuilt or Custom NanoBiT® Assays
Get started with NanoBiT® PPI Assays in a format that suits your needs. Design your own assays using the NanoBiT® Starter Systems, or choose from prebuilt, optimized assays for a number of popular research targets including our newest collection of RAS pathway interaction assays. You can consult with us to design a customized assay specific to your research.
Protocols
Specifications
Catalog Number:
What's in the box
| Item | Part # | Choose a size |
|---|---|---|
pBiT1.1-C [TK/LgBiT] Vector |
N196A | 1 × 20μg |
pBiT2.1-C [TK/SmBiT] Vector |
N197A | 1 × 20μg |
pBiT1.1-N [TK/LgBiT] Vector |
N198A | 1 × 20μg |
pBiT2.1-N [TK/SmBiT] Vector |
N199A | 1 × 20μg |
NanoBiT® Negative Control Vector |
N202A | 1 × 20μg |
LgBiT-PRKAR2A Control Vector |
N203A | 1 × 20μg |
SmBiT-PRKACA Control Vector |
N204A | 1 × 20μg |
Nano-Glo® Live Cell Substrate |
N205A | 2 × 125μl |
Nano-Glo® LCS Dilution Buffer |
N206A | 2 × 2.5ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
See Protocol for detailed storage recommendations.
BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
No transfer of this product is allowed. Researchers may use this product in their own research and they may transfer derivatives to others for research use provided that at the time of transfer a copy of this label license is given to recipients and recipients agree to be bound by the terms of this label license.
Researchers shall have no right to modify or otherwise create variations of the nucleotide sequences of the subunits of NanoBiT® Technology, except that researchers may create fused gene sequences at the termini of the subunits of NanoBiT® Technology. No other use of this product or its derivatives is authorized without the prior express written consent of Promega.
In addition, researchers must either:
(1a) use Nano-Glo®-branded luminescent assay reagents (LARs) for all determinations of luminescence activity of this product and its derivatives, or
(1b) contact Promega to obtain a license for use of the product and its derivatives with LARs not manufactured by Promega.
For uses of Nano-Glo®-branded LARs intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must either:
(2a) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product and its derivatives, or
(2b) contact Promega to obtain a license for use of the product and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega.
Researcher has no right to modify, derivatize, genetically engineer or otherwise create variations of the NanoBiT® Control Vectors, except that researcher may propagate and store NanoBiT® Control Vectors for use in NanoBiT® assays. With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
Researcher may use this product for research use only; no commercial use is allowed. Commercial use means any and all uses of this product by a party in exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the product, whether or not such product is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the product. No other use or transfer of this product is authorized without the prior express written consent of Promega.
For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must:
(i) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(ii) contact Promega to obtain a license for use of the product for energy transfer assays to energy acceptors not manufactured by Promega.
With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
U.S. Pat. No. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.
U.S. Pat. Nos. 9,797,889, 9,797,890 and 11,493,504; European Pat. No. 2970412; Japanese Pat. Nos. 7280842 and 7532562; and other patents pending.
What's in the box
| Item | Part # | Choose a size |
|---|---|---|
|
pF4A CMV Flexi® Vector |
C848A | 1 × 20μg |
pFC34K LgBiT TK-neo Flexi® Vector |
N192A | 1 × 20μg |
pFC36K SmBiT TK-neo Flexi® Vector |
N193A | 1 × 20μg |
pFN33K LgBiT TK-neo Flexi® Vector |
N194A | 1 × 20μg |
pFN35K SmBiT TK-neo Flexi® Vector |
N195A | 1 × 20μg |
NanoBiT® Negative Control Vector |
N202A | 1 × 20μg |
LgBiT-PRKAR2A Control Vector |
N203A | 1 × 20μg |
SmBiT-PRKACA Control Vector |
N204A | 1 × 20μg |
Nano-Glo® Live Cell Substrate |
N205A | 2 × 125μl |
Nano-Glo® LCS Dilution Buffer |
N206A | 2 × 2.5ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
See Protocol for detailed storage recommendations.
BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
No transfer of this product is allowed. Researchers may use this product in their own research and they may transfer derivatives to others for research use provided that at the time of transfer a copy of this label license is given to recipients and recipients agree to be bound by the terms of this label license.
Researchers shall have no right to modify or otherwise create variations of the nucleotide sequences of the subunits of NanoBiT® Technology, except that researchers may create fused gene sequences at the termini of the subunits of NanoBiT® Technology. No other use of this product or its derivatives is authorized without the prior express written consent of Promega.
In addition, researchers must either:
(1a) use Nano-Glo®-branded luminescent assay reagents (LARs) for all determinations of luminescence activity of this product and its derivatives, or
(1b) contact Promega to obtain a license for use of the product and its derivatives with LARs not manufactured by Promega.
For uses of Nano-Glo®-branded LARs intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must either:
(2a) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product and its derivatives, or
(2b) contact Promega to obtain a license for use of the product and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega.
Researcher has no right to modify, derivatize, genetically engineer or otherwise create variations of the NanoBiT® Control Vectors, except that researcher may propagate and store NanoBiT® Control Vectors for use in NanoBiT® assays. With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
Researcher may use this product for research use only; no commercial use is allowed. Commercial use means any and all uses of this product by a party in exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the product, whether or not such product is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the product. No other use or transfer of this product is authorized without the prior express written consent of Promega.
For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must:
(i) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(ii) contact Promega to obtain a license for use of the product for energy transfer assays to energy acceptors not manufactured by Promega.
With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
U.S. Pat. No. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.
What's in the box
| Item | Part # | Choose a size |
|---|---|---|
FRB-LgBiT Control Vector |
N200A | 1 × 20μg |
FKBP-SmBiT Control Vector |
N201A | 1 × 20μg |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
See Protocol for detailed storage recommendations.
BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
No transfer of this product is allowed. Researchers may use this product in their own research and they may transfer derivatives to others for research use provided that at the time of transfer a copy of this label license is given to recipients and recipients agree to be bound by the terms of this label license.
Researchers shall have no right to modify or otherwise create variations of the nucleotide sequences of the subunits of NanoBiT® Technology, except that researchers may create fused gene sequences at the termini of the subunits of NanoBiT® Technology. No other use of this product or its derivatives is authorized without the prior express written consent of Promega.
In addition, researchers must either:
(1a) use Nano-Glo®-branded luminescent assay reagents (LARs) for all determinations of luminescence activity of this product and its derivatives, or
(1b) contact Promega to obtain a license for use of the product and its derivatives with LARs not manufactured by Promega.
For uses of Nano-Glo®-branded LARs intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must either:
(2a) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product and its derivatives, or
(2b) contact Promega to obtain a license for use of the product and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega.
Researcher has no right to modify, derivatize, genetically engineer or otherwise create variations of the NanoBiT® Control Vectors, except that researcher may propagate and store NanoBiT® Control Vectors for use in NanoBiT® assays. With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
U.S. Pat. Nos. 9,797,889, 9,797,890 and 11,493,504; European Pat. No. 2970412; Japanese Pat. Nos. 7280842 and 7532562; and other patents pending.
Resources
Articles
- Using the NanoBiT® PPI System with GloMax® Discover to Detect Protein-Protein Interactions
- Rapid quantification of C. difficile glutamate dehydrogenase and toxin B (TcdB) with a NanoBiT split-luciferase assay
- An interaction between SLC35A1 and ST3Gal4 is differentially affected by CDG-causing mutations in the SLC35A1 gene
- Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.
- In vitro structure–activity relationships and forensic case series of emerging 2‑benzylbenzimidazole ‘nitazene’ opioids
- Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity
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N2011, N2012, N2013
FuGENE® 6 Transfection Reagent
Proven, dependable performance. Best for everyday use with common cell lines.
E2691, E2692, E2693
GloMax® Discover System
High-performance microplate reader for detecting luminescence, fluorescence and absorbance.
GM3000
Anti-LgBiT Monoclonal Antibody
Antibody used to detect NanoBiT® Large BiT (LgBiT) fragment and LgBiT fusion proteins by Western blotting and immunofluorescence.
N7100
