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NanoBRET™ Proteasomal Recruitment Starter Kit

ND2730_NanoBRET--Proteasomal-Recruitment-Starter-Kit_3

Live-Cell BRET-Based Assay to Measure Protein Recruitment to the 26S Proteasome

  • Detect target protein recruitment to the proteasome
  • Perform endpoint or live-cell kinetic analysis of proteasome trafficking
  • Assay proteasome recruitment of ectopic or endogenously expressed proteins

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Catalog number selected: ND2730

$ 1,800.00
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NanoBRET™ Proteasomal Recruitment Starter Kit
1 each
$ 1,800.00
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Assess Proteasomal Recruitment in Live Cells

Regulating overall cellular protein homeostasis is critical for maintaining cell health and often found to be altered by cellular treatments or disease states. The majority of eukaryotic proteins and their abundance is regulated via the ubiquitin proteasome system (UPS), where ubiquitin is conjugated to signal proteins and sends the ubiquitinated complex to the 26S proteasome for degradation.

The NanoBRET™ Proteasomal Recruitment Starter Kit provides the tools to create target-specific live-cell assays to globally monitor recruitment to the 26S proteasome. This assay can be used to study dynamic increases or decreases in 26S trafficking after cellular treatments, such as small molecules or pathway inducers, that would influence protein stabilization or degradation. These 26S proteasome assays can be particularly useful for the study of targeted degradation compounds because the NanoBRET™ signal ratio means you can investigate proteasome recruitment while simultaneously monitoring target protein levels to assess degradation.

NanoBRET™ Assay Principle

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Proteasomal Recruitment Assay Overview

Proteasome Recruitment Assay Overview

How to Get Started

  1. Create the bioluminescent donor protein, which is the target being monitored for proteasome recruitment. 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®-PSMD3 (26S proteasome regulatory subunit 3) fusion protein via transient transfection either 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 proteasome recruitment.
  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. Trafficking to the proteasome will result in energy transfer between the donor and acceptor and an increased NanoBRET™ signal.

Measure Compound-Dependent Levels of Target Protein Recruitment to the Proteasome in a Live-Cell Kinetic Assay

Increased protein trafficking to the proteasome 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 increase target protein ubiquitination, leading to increased trafficking to the 26S proteasome. Here we transiently transfected NanoLuc®-BRD4 and HaloTag®-PSMD3 Fusion Vectors in HEK293 cells, and the NanoBRET™ signal showed the expected kinetic increase in proteasome trafficking when MZ1, a VHL-mediated PROTAC, was added. Assay specificity was demonstrated by adding increasing levels of VH298, which binds VHL and blocks the PROTAC-mediated ternary complex and BRD4 ubiquitination.

Proteasomal recruitment kinetics for BRD4
Monitoring decreases in proteasomal recruitment after signaling pathway modulation

In the absence of Wnt signaling, β‑catenin is phosphorylated and degraded via the UPS, resulting in a high basal level of β‑catenin at the proteasome. Here we use transiently transfected NanoLuc®-β-catenin and HaloTag®-PSMD3 fusion proteins to demonstrate that inhibiting the upstream kinase, GSK‑3β, by treatment with the compound AZD2858 decreased β‑catenin proteasomal recruitment, resulting in increased protein levels. Because β-catenin is the donor protein in the NanoBRET™ assay, its protein level is monitored separately so both relative target protein abundance and proteasome recruitment can be determined in a single assay.

Measure proteasomal recruitment and protein levels

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®-PSMD3 Fusion Vector

N270A 1 × 20μg1μg/μl

SDS

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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 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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