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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, allows analysis of protein association and disassociation
  • Improved sensitivity and accuracy over split firefly luciferase

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NanoBiT® PPI Starter Systems
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NanoBit_OverviewImagery

Protein Interaction Assays that Reflect Natural Cellular Biology

NanoLuc® Binary Technology (NanoBiT) is a structural complementation reporter designed for protein:protein interaction (PPI) studies. The NanoBiT® system is composed of two small subunits, Large BiT (LgBiT; 18kDa) and Small BiT (SmBiT; 11 amino acid peptide), that are expressed as fusions to target proteins of interest. The LgBiT and SmBiT subunits have been optimized for stability and minimal self-association. When two proteins of interest are tagged with these subunits and then interact, the subunits come together to form an active enzyme and generate a bright luminescent signal.  

The small subunit size of the NanoBiT complementation partners minimizes interference with normal protein function, and the bright signal accommodates low, native expression levels.

 

 

NanoBit_Protein interactions technology Paper_1030x1450

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.


Read the Paper

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. 

Download the Poster
NanoBit_ScientificPoster
NanoBiT-video-image

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_XenopusChalkTalk_PosterImage_1280x720
NanoBit_ComplementationChalkTalk_PosterImage_1280x720
NanoBit_Fission_PosterImage_1280x720

NanoBiT® System Vectors

The NanoBiT® PPI Starter Systems provide the vectors required to create LgBiT and SmBiT protein fusions, along with control vectors and the Nano-Glo® Live Cell Assay System, a single-addition, non-lytic detection reagent used for monitoring NanoBiT® luminescence in living cells. See below for details of the vectors provided with each system.
NanoBiT® PPI MCS Starter 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. 

13374MA-W
pBiT1.1-N[TK/LgBiT] Vector sequence text file
13373MA-W
pBiT2.1-C[TK/SmBiT] Vector sequence text file.
13369MA-W
SmBiT-PRKACA Control Vector sequence text file.
13372MA-W
pBiT1.1-C[TK/LgBiT] Vector sequence text file.
13367MA-W
NanoBiT® Negative Control Vector sequence text file.
13375MA-W
pBiT2.1-N[TK/SmBiT] Vector sequence text file.
13368MA-W
LgBiT-PRKAR2A Control Vector sequence text file.
NanoBiT® PPI Flexi® Starter System Vectors

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.

13370MA-W
pFN33K LgBiT TK-neo Flexi® Vector sequence text file.
13364MA-W
pFC36K SmBiT TK-Neo Flexi® Vector sequence text file.
13363MA-W
pFC34K LgBiT TK-Neo Flexi® Vector sequence text file.
13367MA-W
NanoBiT® Negative Control Vector sequence text file.
13371MA-W
pFN35K SmBiT TK-neo Flexi® Vector sequence text file.
13368MA-W
LgBiT-PRKAR2A Control Vector sequence text file.
13369MA-W
SmBiT-PRKACA Control Vector sequence text file.
pF4A CMV Flexi Vector.
pF4A CMV Flexi® Vector Accession Number AY753580; sequence text file.
NanoBiT® PPI Control  Pair (FKPB, FRB)

The NanoBiT® PPI Control Pair (FKBP:FRB; Cat.# N2016) is available separately as an inducible positive control.  

13365MA-W
FRB-LgBit Control Vector sequence text file.
13366MA-W
FKBP-SmBiT Control Vector sequence text file.
Custom NanoBiT® Vectors
NanoBiT® Vectors with a CMV promoter are available by special order from Promega Custom Assay Services.

Protocols

Specifications

You are viewing: 1 each Change Configuration

What's in the box?

Item Part # Size

pBiT1.1-C [TK/LgBiT] Vector

N196A 1 x 20μg

pBiT2.1-C [TK/SmBiT] Vector

N197A 1 x 20μg

pBiT1.1-N [TK/LgBiT] Vector

N198A 1 x 20μg

pBiT2.1-N [TK/SmBiT] Vector

N199A 1 x 20μg

NanoBiT® Negative Control Vector

N202A 1 x 20μg

LgBiT-PRKAR2A Control Vector

N203A 1 x 20μg

SmBiT-PRKACA Control Vector

N204A 1 x 20μg

Nano-Glo® Live Cell Substrate

N205A 2 x 125μl

Nano-Glo® LCS Dilution Buffer

N206A 2 x 2.5ml

SDS

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Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

See Protocol for detailed storage recommendations.

Patents and Disclaimers

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:
(a) use NanoBRET™-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(b) 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.

Patent Pending.

U.S. Pat. No. 8,809,529 and other patents pending.

Specifications

You are viewing: 1 each Change Configuration

What's in the box?

Item Part # Size

pF4A CMV Flexi® Vector

C848A 1 x 20μg

pFC34K LgBiT TK-neo Flexi® Vector

N192A 1 x 20μg

pFC36K SmBiT TK-neo Flexi® Vector

N193A 1 x 20μg

pFN33K LgBiT TK-neo Flexi® Vector

N194A 1 x 20μg

pFN35K SmBiT TK-neo Flexi® Vector

N195A 1 x 20μg

NanoBiT® Negative Control Vector

N202A 1 x 20μg

LgBiT-PRKAR2A Control Vector

N203A 1 x 20μg

SmBiT-PRKACA Control Vector

N204A 1 x 20μg

Nano-Glo® Live Cell Substrate

N205A 2 x 125μl

Nano-Glo® LCS Dilution Buffer

N206A 2 x 2.5ml

SDS

Choose language:

Certificate of Analysis

Search for Specific Certificate:

View more results
No results
Loading…

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

See Protocol for detailed storage recommendations.

Patents and Disclaimers

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:
(a) use NanoBRET™-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(b) 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.

Patent Pending.

U.S. Pat. No. 8,809,529 and other patents pending.

Specifications

You are viewing: 1 each Change Configuration

What's in the box?

Item Part # Size

FRB-LgBiT Control Vector

N200A 1 x 20μg

FKBP-SmBiT Control Vector

N201A 1 x 20μg

SDS

Choose language:

Certificate of Analysis

Search for Specific Certificate:

View more results
No results
Loading…

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

See Protocol for detailed storage recommendations.

Patents and Disclaimers

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.

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