LgBiT Expression Vector and Stable Cell Line
Express Intracellular LgBiT Protein for HiBiT Analysis
- Perform live-cell, kinetic analysis of HiBiT-tagged proteins; no cell lysis required
- Compatible with exogenously or endogenously expressed HiBiT fusion proteins
- Use complemented HiBiT as the energy donor for live-cell NanoBRET® applications
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Catalog Number: N2681
Catalog Number: N2672
Study Protein Dynamics under Endogenous Expression Conditions
The HEK293 LgBiT Cell Line and LgBiT Expression Vector are reagents for constitutive intracellular expression of the LgBiT protein, which has a high-affinity interaction with the 11-amino-acid HiBiT tag to reconstitute the NanoBiT® enzyme. These intracellular LgBiT expression tools can be paired with HiBiT-tagged proteins expressed either transiently, stably or endogenously via CRISPR-Cas9 insertion for endpoint or kinetic real-time monitoring of HiBiT protein levels without cell lysis. Once HiBiT is expressed, NanoBiT® enzyme activity is detected with one of the Nano-Glo® Live Cell Substrates, which provide options that balance brightness and signal stability.
Intracellular LgBiT expression expands the HiBiT tag use to live-cell applications such as measuring real-time protein dynamics in disease models or after treating cells with targeted protein degraders, developing viral infectivity models or assays to monitor intracellular delivery of peptides or small molecules, and using HiBiT-tagged proteins as the energy donor in NanoBRET® interaction assays.
Measure Real-Time Abundance of Endogenously Regulated HiBiT-Tagged Proteins
Perform quantitative time-course studies on the same sample by adding a single reagent and simply measuring bioluminescence over time. No need to collect multiple samples, guess at which endpoints to measure, or develop antibodies against target proteins. There are several options for incorporating intracellular LgBiT into your CRISPR-Cas9 HiBiT knock-in workflow:
- Use the HEK293 LgBiT Cell Line to create HiBiT-knock-in cell pools or clones. Additional LgBiT stable cell lines are also available through Promega custom assay services.
- Create a stable LgBiT cell line of your choice using the LgBiT Expression Vector and further edit the cell line to knock-in HiBiT.
- Edit HiBiT into the endogenous locus of your cell line of choice and create a stable cell line. Transfect the LgBiT Expression Vector to either create a stable LgBiT-expressing variant or transiently express the LgBiT protein.
Live-Cell, Kinetic Monitoring of Endogenous HiBiT-BRD4 Following PROTAC Treatment
HEK293 LgBiT Cell Line CRISPR-Cas9 Edited to Knock-In HiBiT
Transient LgBiT Expression Following HiBiT Knock-In into HEK293 Cells
Use Complemented HiBiT as the Energy Donor in NanoBRET® Applications for More Assay Possibilities
The intracellular LgBiT expression reagents allow HiBiT-tagged proteins to be used as the bioluminescent energy donor in live-cell NanoBRET® assays for the study of protein:protein interactions or small molecule target engagement. NanoBRET® technology is a proximity-based method dependent upon energy transfer from a bioluminescent energy donor protein to a fluorescent acceptor. Live-cell HiBiT complementation creates the bioluminescent donor, which can be coupled with different fluorescent acceptors to create live-cell interactions assays with HiBiT-tagged protein.
Ubiquitination Kinetics of Endogenously Expressed HiBiT-BRD4
HEK293 LgBiT Stable Cell Line was modified using CRISPR-Cas9 gene editing to create cells expressing a HiBiT-BRD4 fusion at the endogenous BRD4 locus. BRD4 ubiquitination levels were monitored following treatment with various PROTAC compounds by transiently transfecting the HaloTag®-Ubiquitin Fusion Vector. Energy transfer from the bioluminescent BRD4 donor to HaloTag®-Ubiquitin fluorescent acceptor was measured using the NanoBRET® Nano-Glo® Kinetic Detection System.
Protocols
Specifications
Catalog Number:
Contenido
| Item | Part # | Presentación | Concentración |
|---|---|---|---|
LgBiT Expression Vector |
N268A | 1 × 20μg | 1μg/μl |
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Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Condiciones de Almacenaje
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. No. 9,797,889 and other patents pending.
Contenido
| Item | Part # | Presentación |
|---|---|---|
HEK293 LgBiT Stable Cell Line |
N267A | 2 × 1ml |
SDS
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Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Condiciones de Almacenaje
LgBiT Stable Cell Line Limited Use Label License
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, which may include a) drug discovery and development and b) the right to modify, derivatize, genetically engineer or otherwise create variations of the product (“derivatives”). No transfer of this product is allowed; however, researcher may transfer derivatives to others provided that at the time of transfer a copy of this label license is given to recipient and recipient agrees to be bound by the terms of this label license. No commercial use of this product or derivatives is allowed. Commercial use means any and all uses of this product or derivatives 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 or its derivatives, whether or not such product or derivatives are resold for use in research. Researcher shall have no right to modify or otherwise create variations of the nucleotide sequences of the subunits of NanoBiT® Technology.
In addition, researcher must either:
(1a) use Nano-Glo®-branded luminescent assay reagents (LARs) for all determinations of luminescence activity of this product, copies or derivatives thereof; or
(1b) contact Promega to obtain a license for use of the product, copies or 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, researcher must:
(2a) use NanoBRET™-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product, copies or derivatives thereof; or
(2b) contact Promega to obtain a license for use of the product, copies or derivatives 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. 9,797,889 and other patents pending.
HEK293 cells were obtained under license from AdVec Inc.
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