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NanoBRET™ Ubiquitination Starter Kit

ND2690_NanoBRET--Ubiquitination-Starter-Kit_3

Live-Cell BRET-Based Assay to Measure Protein Ubiquitination

  • General ubiquitination assay to measure changes in the relative level of target protein ubiquitination
  • Perform endpoint or live-cell kinetic analysis to determine protein ubiquitination dynamics
  • Assay ubiquitination on ectopic or endogenously expressed proteins

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Catalog number selected: ND2690

$ 1,800.00
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NanoBRET™ Ubiquitination Starter Kit
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Measure Target Protein Ubiquitination in Live Cells

Regulating overall cellular protein homeostasis is critical for maintaining cell health and often altered by cellular treatments or disease states. Most proteins and their abundance are regulated via the ubiquitin proteasome system (UPS), which uses ubiquitin conjugation to signal proteins to be trafficked to the proteasome for degradation. Ubiquitination on any given target can vary in levels, mono- and poly-ubiquitination, and mediated through a variety of amino acid linkages.

The NanoBRET™ Ubiquitination Starter Kit provides the tools to create target-specific live-cell ubiquitin assays that globally measure all types of ubiquitination on a target protein. The assays can be used to measure dynamic increases or decreases in the relative levels of target protein ubiquitination following cellular treatments, such as small molecules or pathway inducers, that would influence protein stabilization or degradation, respectively. These assays can be particularly useful for studying targeted degradation compounds because the bioluminescent resonance energy transfer ratio of the NanoBRET™ signal means you can investigate protein ubiquitination while simultaneously monitoring target protein levels to assess degradation.

NanoBRET™ Assay Principle

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Ubiquitination Assay Overview

Ubiquitination BRET Assay Overview

How to Get Started

  1. Create the bioluminescent donor protein that is the ubiquitination target. The donor protein can be created as a NanoLuc® fusion protein with the provided NanoLuc® cloning vectors or an endogenous target tagged with HiBiT with 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 HaloTag®-Ubiquitin fusion proteins via transient transfection either by co-transfecting with the NanoLuc® fusion protein or with 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 compounds that affect the target protein ubiquitination levels.
  4. Add the NanoBRET™ Nano-Glo® reagents, either endpoint or kinetic, that 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.

Measure Compound-Dependent Levels of Target Protein Ubiquitination in a Live-Cell Assay

Detecting protein ubiquitination kinetics in response to degrader compound treatment

Targeted degradation compounds, such as PROTACs and molecular glues, create a ternary complex between the protein and an E3 ligase component. A productive and active ternary complex will result in increased target protein ubiquitination and subsequent degradation via the UPS. Here we show an endogenously tagged protein, HiBiT-BRD4, with varying kinetic increases in ubiquitination levels that depend on specific PROTAC treatment.

Endogenous BRD4 ubiquitination kinetics
Monitoring changes in ubiquitination and multiplexed target protein levels after signaling pathway modulation

In the absence of Wnt signaling, β-catenin is phosphorylated and degraded, resulting in a high level of ubiquitinated β-catenin. Here we use endogenous HiBiT-tagged β-catenin and transiently transfected HaloTag®-Ubiquitin Fusion and LgBiT Vectors to demonstrate that inhibiting the upstream kinase, GSK-3β, by treatment with the compound AZD2858 results in decreased β-catenin ubiquitination and subsequent protein stabilization. Since β-catenin is the donor protein in the NanoBRET™ assay, its protein level is monitored separately in the same assay. Therefore, you can determine relative abundance of the target protein and ubiquitination in a single assay.

Monitoring changes in ubiquitination and multiplexed target protein levels after signaling pathway modulation

Specifications

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®-Ubiquitin Fusion Vector

N272A 1 × 20μg1μg/μl

SDS

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Download SDSPDF (372 KB) – English (United States)

Certificate of Analysis

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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, 9,951,373 and 10,633,690 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.

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