NanoBiT® BiBiT PPI Starter System

1種類のプラスミドで簡便に：再現性を高めたNanoBiT®アッセイ

単一ベクター設計による安定発現細胞株の作製の簡便化
リアルタイム発光によるライブセルでの動的なPPI検出
高いS/B比による高感度かつ再現性の高い結果、HTSへのスケール展開も容易
創薬スクリーニング、シグナル伝達、バイオロジクス研究に最適

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単一ベクターで簡便・高感度に実現するタンパク質相互作用アッセイ

NanoBiT® BiBiT PPI Starter System は、NanoBiT® (NanoLuc® Binary Technology) ルシフェラーゼ相補法を利用しており、LgBiTおよびSmBiTサブユニットを用いて、生細胞内でのタンパク質間相互作用（PPI）を発光でモニタリングします。低親和性かつ可逆的な酵素補完により、PPIの形成だけでなく、その解離も動的に観察することが可能です。

BiBiT対応ベクターは、柔軟なタグ付け構成をサポートしており、単一のベクターから一過性発現または安定発現のいずれにも対応できます。特に本システムでは、初期のTKプロモーター版とは異なり、CMVプロモーターを採用しているため、さまざまな細胞株において高い発現レベルが得られます。

本システムはアッセイのセットアップを簡素化し、再現性を向上させるため、創薬スクリーニング、シグナル伝達経路の解析、機能的PPI研究に最適です。

BiBiTによる安定発現細胞株の構築

Schematic of the BiBiT approach to stable cell line development.

最大8種類のBiBiT対応ベクターを作製し、目的のタンパク質に対する最適なタグ付け方向をスクリーニング・見出すことができます。最も高いシグナル/バックグラウンド比（S/B比）を示すBiBiT対応ベクターペアは、1つのBiBiTベクターにライゲーションすることで、双方向性CMVプロモーターにより両融合タンパク質を安定かつバランスよく発現させることができます。ブラスチシジン耐性遺伝子により、哺乳類細胞における安定的な導入イベントの選択が可能です。

BiBiT単一ベクターアプローチの利点

同一座位から両融合タンパク質を同程度に共発現
実験間のばらつきの低減
S/B比（シグナル/バックグラウンド比）の向上
アッセイ感度の強化

BiBiTを用いた安定発現細胞株でアッセイの堅牢性を向上

BiBiTベクターから安定発現細胞株を樹立することで、NanoBiT®アッセイにおけるばらつきが低減され、特にハイスループットスクリーニング環境下での信頼性が向上します。

従来の一過性発現法と比較して、BiBiTを用いた安定発現系は以下の利点があります：

アッセイの精度向上
シグナル/バックグラウンド比の改善
Z′値の上昇による、信頼性の高い化合物プロファイリングとスクリーニングの実現

	従来のNanoBiT® 一過性発現	NanoBiT® BiBiT 安定発現
IC₅₀ (nM)	238	343
SB	11	19
SN	11	83
Z′	0.68	0.96

一過性発現とBiBiTベースの安定発現を用いたCRAF/BRAF NanoBiT® アッセイの性能比較　安定発現細胞株ではZ′値およびアッセイウィンドウが改善されており、アッセイ品質の向上とばらつきの低減を示している。

抗体のPPI制御作用を定量化

Line graph showing dose-dependent inhibition of HER2:HER3 interaction by pertuzumab detected using the BiBiT system.

Line graph showing dose-dependent inhibition of HER2:HER3 interaction by trastuzumab detected using the BiBiT system.

Line graph showing dose-dependent inhibition of HER2:HER3 interaction by rituximab detected using the BiBiT system.

NanoBiT® BiBiT PPI Starter System は、治療用抗体に応答したHER2:HER3相互作用を高感度かつ定量的に測定可能である　NanoBiTタグ付きHER2およびHER3を安定発現する細胞株を用いることで、パートゥズマブ、トラスツズマブ、リツキシマブに対する用量依存的な応答の違いが観察された。これらの結果は、NanoBiT® PPIアッセイが受容体結合に対する抗体の機能的影響を識別できることを示しており、生理学的に関連性の高い条件下での創薬スクリーニングおよび候補抗体の特性評価に適したプラットフォームであることを示している。

ニーズに合わせて選べるスターターシステム、既製アッセイ、カスタム設計

NanoBiT® PPI アッセイは、目的や研究スタイルに応じた形式で導入できます。NanoBiT® Starter System を使って独自のアッセイを設計することも、最適化された既製のアッセイを選ぶことも可能です。既製のアッセイには、最新のRAS経路に関する相互作用アッセイを含む、人気の研究ターゲットが多数揃っています。研究内容に合わせて、カスタムアッセイの設計についてご相談いただくことも可能です。

既成のアッセイを見るカスタムアッセイのお問合せ

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保存条件

パテントおよび免責条項

Promega DNA Vector Limited Use Label License (LULL)

BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.

1. Usage Restrictions:
- 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation.
- 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including but not limited to product manufacture or resale of the material for any use and sale of derivatives for any use.
- 1.3. If recipient is a contract research organization (“CRO”), recipient may transfer or sell data or information generated using the material or derivatives to CRO’s customers who request such data or information, provided that the material or derivatives cannot be transferred from CRO to CRO’s customers.
2. Modification Restrictions:
- 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors.
- 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes.
3. For determinations of luminescence activity of this material and its derivatives, recipient must either:
- 3.1. Use Promega-branded luminescent assay reagents (LARs); or
- 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either:
- 4.1. Use Nano-Glo®-branded LARs; or
- 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either:
- 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or
- 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must:
- 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and
- 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or
- 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein.
7. For uses of HaloTag® Technology in this material, recipient must either:
- 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or
- 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.
8. Transfer of Materials:
- 8.1. Unmodified material or progeny of such material cannot be transferred.
- 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license.
- 8.3. If recipient is not a CRO, user may transfer this material to a CRO in order to have CRO use the material solely on behalf of recipient to provide recipient data or information, and CRO may transfer or sell such data or information to recipient, provided that recipient provides CRO with a copy of this label license and CRO agrees to comply with this label license.
- 8.4. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party.
9. Other Uses:
- 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information.
10. Disclaimer and Governing Law:
- 10.1. 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 MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

SDS

Search for SDS

分析証明書

Search by lot number

保存条件

パテントおよび免責条項

Promega DNA Vector Limited Use Label License (LULL)

BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.

1. Usage Restrictions:
- 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation.
- 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including but not limited to product manufacture or resale of the material for any use and sale of derivatives for any use.
- 1.3. If recipient is a contract research organization (“CRO”), recipient may transfer or sell data or information generated using the material or derivatives to CRO’s customers who request such data or information, provided that the material or derivatives cannot be transferred from CRO to CRO’s customers.
2. Modification Restrictions:
- 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors.
- 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes.
3. For determinations of luminescence activity of this material and its derivatives, recipient must either:
- 3.1. Use Promega-branded luminescent assay reagents (LARs); or
- 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either:
- 4.1. Use Nano-Glo®-branded LARs; or
- 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either:
- 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or
- 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must:
- 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and
- 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or
- 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein.
7. For uses of HaloTag® Technology in this material, recipient must either:
- 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or
- 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.
8. Transfer of Materials:
- 8.1. Unmodified material or progeny of such material cannot be transferred.
- 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license.
- 8.3. If recipient is not a CRO, user may transfer this material to a CRO in order to have CRO use the material solely on behalf of recipient to provide recipient data or information, and CRO may transfer or sell such data or information to recipient, provided that recipient provides CRO with a copy of this label license and CRO agrees to comply with this label license.
- 8.4. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party.
9. Other Uses:
- 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information.
10. Disclaimer and Governing Law:
- 10.1. 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 MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

SDS

Search for SDS

分析証明書

Search by lot number

保存条件

パテントおよび免責条項

Promega DNA Vector Limited Use Label License (LULL)

BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.

1. Usage Restrictions:
- 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation.
- 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including but not limited to product manufacture or resale of the material for any use and sale of derivatives for any use.
- 1.3. If recipient is a contract research organization (“CRO”), recipient may transfer or sell data or information generated using the material or derivatives to CRO’s customers who request such data or information, provided that the material or derivatives cannot be transferred from CRO to CRO’s customers.
2. Modification Restrictions:
- 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors.
- 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes.
3. For determinations of luminescence activity of this material and its derivatives, recipient must either:
- 3.1. Use Promega-branded luminescent assay reagents (LARs); or
- 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either:
- 4.1. Use Nano-Glo®-branded LARs; or
- 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either:
- 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or
- 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must:
- 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and
- 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or
- 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein.
7. For uses of HaloTag® Technology in this material, recipient must either:
- 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or
- 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.
8. Transfer of Materials:
- 8.1. Unmodified material or progeny of such material cannot be transferred.
- 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license.
- 8.3. If recipient is not a CRO, user may transfer this material to a CRO in order to have CRO use the material solely on behalf of recipient to provide recipient data or information, and CRO may transfer or sell such data or information to recipient, provided that recipient provides CRO with a copy of this label license and CRO agrees to comply with this label license.
- 8.4. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party.
9. Other Uses:
- 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information.
10. Disclaimer and Governing Law:
- 10.1. 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 MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

技術資料

注目の技術資料

実証されたBiBiTアッセイの性能

安定発現細胞株の構築におけるBiBiTアプローチの詳細は、ポスター資料をご覧ください。本資料では、1つのゲノム座位からLgBiTおよびSmBiT融合タンパク質を安定かつバランスよく発現させるためのBiBiT対応ベクターの活用法を紹介しています。

掲載事例として、HER2:HER3、VEGFR1:VEGFR1、CX3CR1:ARRB2、CRAF:BRAFなど、重要な膜タンパク質間相互作用を取り上げています。この堅牢なアプローチは、アッセイの再現性と感度を最大限に高めることができます。

ポスターを見る

novel-cloning-vectors-for-stable-expression-of-nanobit-fusion-proteins-gpn067

NanoBiT® BiBiT PPI Starter System

1種類のプラスミドで簡便に：再現性を高めたNanoBiT®アッセイ

単一ベクターで簡便・高感度に実現するタンパク質相互作用アッセイ