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NanoBRET™ VHL and CRBN Ternary Complex Starter Kits

Live-Cell BRET-Based Assays to Measure Compound-Induced E3 Ligase Ternary Complex Formation

  • Measure induced interaction between target protein and E3 ligase components VHL or CRBN with degrader compound treatment
  • Simultaneously monitor target protein levels in the presence of degrader
  • Perform endpoint or live-cell kinetic analysis of ternary complex formation

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Catalog number selected: ND2700

$ 1,800.00
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NanoBRET™ VHL and CRBN Ternary Complex Starter Kits
VHL/1 each
$ 1,800.00
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Investigate Ternary Complex Formation

Targeted degradation via small molecule degraders such as PROTACs or molecular glues require forming a ternary complex that consists of target protein:degrader compound:E3 ligase component. Ternary complex formation is the first mechanistic step required for target ubiquitination and degradation via the ubiquitin proteasome pathway, representing a key step in optimizing effective degrader compounds. The NanoBRET™ Ternary Complex Starter Kits provide the tools to create target-specific assays for live-cell detection of complex formation with either the VHL (von Hippel-Lindau disease tumor suppressor) or CRBN (cereblon) E3 ligase components. These assays use NanoBRET™ technology, a proximity-based method dependent upon energy transfer from a luminescent donor target protein to a fluorescent acceptor and can be performed using either endpoint or kinetic format to monitor the rate and stability of complex formation. The ratio of the bioluminescent and fluorescent signal in the NanoBRET™ assay means you can measure ternary complex formation during target protein degradation and simultaneously monitor target protein levels.

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NanoBRET™ Assay Principle

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Ternary Complex Assay Overview

PROTAC Ternary Complex Formation Assay Overview

How to Get Started

  1. Create the bioluminescent donor protein, which is the target for degradation. The donor protein can be created as a NanoLuc® fusion protein with the provided NanoLuc® cloning vectors or as an endogenous target tagged with HiBiT by CRISPR-Cas9 gene editing. Express the NanoLuc® fusion either transiently or stably in any cell line that can be transfected. Transfect the LgBiT expression vector into HiBiT CRISPR cell lines to produce binary complementation in the cell. The NanoLuc®-BRD4 FL Fusion Vector is provided as a positive control.
  2. Introduce the HaloTag®-VHL or CRBN fusion protein via transient transfection by either co-transfecting with the NanoLuc® fusion protein or the LgBiT vector if using a HiBiT CRISPR cell line. Labeled HaloTag® fusion protein will serve as the fluorescent acceptor in the BRET assay. HaloTag® protein, expressed from the HaloTag® Control Vector, is used as a negative control.
  3. Treat cells with degrader compounds to induce ternary complex formation. If monitoring kinetic ternary complex stability, protein level or both is desired, perform experiments with and without MG132.
  4. Add the NanoBRET™ Nano-Glo® reagents, endpoint or kinetic, which provide the substrate for the donor protein and the fluorescent NanoBRET™ HaloTag® 618 Ligand for the acceptor protein. Measure the donor and acceptor signal using filtered luminescence, and determine NanoBRET™ ratio. Ternary complex formation results in energy transfer between the donor and acceptor, increasing the NanoBRET™ signal.

Monitor Ternary Complex Formation and Target Protein Levels in the Same Assay

NanoBRET™ Ternary Complex Formation Assays are well suited for live-cell monitoring of the ternary complex that forms between the target protein and E3 ligase component when treated with targeted degradation compounds, such as PROTACs and molecular glues. The assay can provide mechanistic understanding of differences in degrader compound efficacy, perform compound optimization studies or confirm mechanism of action for on- and off-target degradation. Here we show NanoBRET™ ternary complex formation for both a PROTAC (NanoLuc®-BRD4:HaloTag®-VHL fusion proteins) and also a molecular glue (NanoLuc®-CK1α:HaloTag®-CRBN fusion proteins) using transiently transfected vectors in HEK293 cells. Since the target being degraded in both assays is the donor protein, its protein level is monitored separately in the same assay. Treatment with the proteasome inhibitor MG132 can be included to increase assay window but omitted when determining the effect on target protein levels.

Measuring ternary complex formation and protein levels

Measure the Kinetics of Ternary Complex Formation

The NanoBRET™ Nano-Glo® Kinetic Detection System provides extended live-cell measurement of ternary complex formation to determine how changes in degradation compound composition will affect the kinetics of ternary complex formation, stability or both in live cells. Here we show ternary complex formation using an endogenously tagged protein, HiBiT-BRD4, with expression of LgBiT and HaloTag®-VHL or HaloTag®-CRBN fusion proteins in HEK293 cells in the presence of MG132. Varying kinetic increases of ternary complex formation are observed with each PROTAC for the same target.

Measure kinetic changes in ternary complex formation

Specifications

You are viewing: ND2700 Change Configuration

What's in the box?

Item Part # SizeConcentrationAvailable Separately

HaloTag® Control Vector

G659A 1 × 20μg

HaloTag® NanoBRET™ 618 Ligand

G980A 1 × 20μl View Product

pNLF1-N [CMV/Hygro] Vector

N135A 1 × 20μg1μg/μl

pNLF1-C [CMV/Hygro] Vector

N136A 1 × 20μg1μg/μl

NanoBRET™ Nano-Glo® Substrate

N157A 1 × 50μl

NanoLuc®-BRD4 FL Fusion Vector

N169A 1 × 20μg

HaloTag®-VHL Fusion Vector

N273A 1 × 20μg1μg/μl

SDS

Choose language:

Download SDSPDF (371 KB) – English (United States)

Use Restrictions

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

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.

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.

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 conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

Researchers may use this product for research use only; no transfer or commercial use of this product 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. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the luciferase gene except that researcher may (1) create fused gene sequences provided that the coding sequence of the resulting luciferase gene has no more than four deoxynucleotides missing at the affected terminus compared to the intact luciferase gene sequence; and (2) insert and remove nucleic acid sequences in splicing research predicated on the inactivation or reconstitution of the luminescence of the encoded luciferase. No other use of this product or derivatives is authorized without the prior express written consent of Promega.

In addition, researchers must:
(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:
(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.

Researchers may transfer derivatives 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. 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 agreement 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 STATEMENT. If the researcher is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the researcher with a full refund. Researchers may use this product for research use only, no commercial use is allowed. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the HaloTag® gene. Researchers may however clone heterologous DNA sequences at either or both ends of said HaloTag® gene so as to create fused gene sequences provided that the coding sequence of the resulting HaloTag® gene has no more than four (4) deoxynucleotides missing at the affected terminus when compared to the intact HaloTag® gene sequence. In addition, researchers must do one of the following in conjunction with use of the product: (1) use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties, or (2) contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. Researchers may transfer derivatives 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. With respect to any uses outside this label license, including any diagnostic, therapeutic or prophylactic 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 REGARDS TO THE PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730 and 9,951,373 and other patents and patents pending.

U.S. Pat. No. 8,809,529, European Pat. No. 2635582 and other patents and patents pending.

U.S. Pat. Nos. 7,425,436, 7,935,803, 8,466,269, 8,742,086, 8,420,367, 8,748,148, 9,416,353, 9,593,316 and other patents and patents pending.

U.S. Pat. Nos. 10,067,149 and 10,024,862 and other patents and patents pending.

Licensed under EP1295121 and EP1088233.

Licensed from Kazusa Genome Technologies.

Specifications

You are viewing: ND2720 Change Configuration

What's in the box?

Item Part # SizeConcentrationAvailable Separately

HaloTag® Control Vector

G659A 1 × 20μg

HaloTag® NanoBRET™ 618 Ligand

G980A 1 × 20μl View Product

pNLF1-N [CMV/Hygro] Vector

N135A 1 × 20μg1μg/μl

pNLF1-C [CMV/Hygro] Vector

N136A 1 × 20μg1μg/μl

NanoBRET™ Nano-Glo® Substrate

N157A 1 × 50μl

NanoLuc®-BRD4 FL Fusion Vector

N169A 1 × 20μg

HaloTag®-CRBN Fusion Vector

N269A 1 × 20μg1μg/μl

SDS

Choose language:

Download SDSPDF (370 KB) – English (United States)

Use Restrictions

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

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.

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.

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 conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

Researchers may use this product for research use only; no transfer or commercial use of this product 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. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the luciferase gene except that researcher may (1) create fused gene sequences provided that the coding sequence of the resulting luciferase gene has no more than four deoxynucleotides missing at the affected terminus compared to the intact luciferase gene sequence; and (2) insert and remove nucleic acid sequences in splicing research predicated on the inactivation or reconstitution of the luminescence of the encoded luciferase. No other use of this product or derivatives is authorized without the prior express written consent of Promega.

In addition, researchers must:
(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:
(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.

Researchers may transfer derivatives 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. 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 agreement 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 STATEMENT. If the researcher is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the researcher with a full refund. Researchers may use this product for research use only, no commercial use is allowed. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the HaloTag® gene. Researchers may however clone heterologous DNA sequences at either or both ends of said HaloTag® gene so as to create fused gene sequences provided that the coding sequence of the resulting HaloTag® gene has no more than four (4) deoxynucleotides missing at the affected terminus when compared to the intact HaloTag® gene sequence. In addition, researchers must do one of the following in conjunction with use of the product: (1) use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties, or (2) contact Promega to obtain a license if Promega HaloTag® ligands are not to be used. Researchers may transfer derivatives 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. With respect to any uses outside this label license, including any diagnostic, therapeutic or prophylactic 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 REGARDS TO THE PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730 and 9,951,373 and other patents and patents pending.

U.S. Pat. No. 8,809,529, European Pat. No. 2635582 and other patents and patents pending.

U.S. Pat. Nos. 7,425,436, 7,935,803, 8,466,269, 8,742,086, 8,420,367, 8,748,148, 9,416,353, 9,593,316 and other patents and patents pending.

U.S. Pat. Nos. 10,067,149 and 10,024,862 and other patents and patents pending.

Licensed under EP1295121 and EP1088233.

Licensed from Kazusa Genome Technologies.

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