HaloPROTAC3

Small Molecule Degrader For Challenging Targets

HaloPROTAC3 is a small-molecule degrader that specifically binds to and degrades HaloTag and its fusion partners in live cells and can be used to study the degradation of targets that would be challenging with target-specific PROTACs. HaloPROTAC3 binds irreversibly to HaloTag and HaloTag® target fusions, recruiting them via co-engagement with an E3 ligase component (VHL) to active E2/E3 ubiquitin ligase complexes and leading to ubiquitination and subsequent degradation by the proteasome.

The ent-HaloPROTAC3 is the enantiomeric compound of HaloPROTAC3 and serves as a negative control. ent-HaloPROTAC3 has the same molecular weight and general structure as HaloPROTAC3 but contains d-hydroxyproline and d-valine residue modifications allowing it to bind HaloTag but not VHL. Use ent-HaloPROTAC3 to confirm that degradation of the HaloTag® fusion protein is mediated through VHL engagement and the PROTAC mechanism.

We also provide a separate NanoLuc®-HaloTag® fusion vector, NanoBRET® Positive Control (Cat.# N1581), that can be used for transient transfection expression and degradation with HaloPROTAC3. See the technical manual for an optional protocol using the luminescence functionality of the NanoBRET® Positive Control to quantify degradation without the use of antibodies.

How HaloPROTAC3 Promotes Target Degradation

HaloPROTAC3 interacts irreversibly with the 34kDa HaloTag® protein through its chloroalkane moiety, forming a ternary complex between the HaloTag® fusion protein and the VHL E3 ligase component. This induced complex promotes the ubiquitination of HaloTag itself and the subsequent degradation of the HaloTag® target fusion via the ubiquitin proteasomal pathway.

HaloPROTAC3 and HaloTag-Edited Cells For Protein Knockout Studies

HaloPROTAC3 can be used to remove endogenous target protein in protein knockout studies. This may require endogenously tagging the target gene with HaloTag using CRISPR/Cas9 editing. To overcome initial low efficiency of HaloTag insertion with a CRISPR pool, use fluorescent Janelia HaloTag® ligands to specifically enrich for HaloTag-edited cells using FACS® sorting.

HaloPROTAC3 removing endogenous target protein in protein knockout studies — Endogenous HaloTag® fusions are created using CRISPR/Cas9 editing with a target-specific crRNA, purified Cas9 and dsDNA plasmid donor vector flanked with 500bp homology arms to the target protein. CRISPR-edited cells can be labeled with the fluorescent HaloTag® ligand, Janelia Fluor® 646 (Cat.# GA1120, GA1121), which will bind to HaloTag® fusions. Enrichment and clone selection of the HaloTag® positive cells from parent cells can be completed with FACS® sorting.

How to Detect HaloTag® Fusion Degradation

You can detect HaloTag® fusion protein by Western blot analysis using antibodies to the target protein or HaloTag itself (left panel) or by pairing the HaloTag with the 11-amino-acid HiBiT tag (right panel). HiBiT binds to LgBiT protein (expressed in cells or added post-lysis) to produce luminescence proportional to target protein levels.

Western Blot

HiBiT Detection

HaloTag® fusion protein detection by pairing with HiBiT — CRISPR/Cas9 genomic insertion of HiBiT-HaloTag to the N-terminus of BRD4 in HEK293 cells stably expressing LgBiT protein. HiBiT-HaloTag-BRD4 cells were treated with various concentrations of HaloPROTAC3 for 3 hours and 24 hours, respectively, and degradation was assessed by measuring luminescence with Nano-Glo® HiBiT Lytic Detection System (Cat.# N3030, N3040, N3050) using a GloMax® Discover (Cat.# GM3000).

HaloPROTAC3 Efficiently Degrades HaloTag® Fusion Protein

This example shows how addition of HaloPROTAC3 results in efficient degradation of HaloTag-HiBiT endogenously tagged β-catenin after Wnt3a stimulation.

Add HaloPROTAC3 for efficient degradation of tagged β-catenin — β-catenin-HaloTag-HiBiT levels increased after Wnt3a stimulation in live HEK293 cells stably expressing LgBiT by tracking HiBiT luminescence over 24 hours using the Nano-Glo® Endurazine Substrate (Cat.# N2570, N2571, N2572). Treatment with 1µM of HaloPROTAC3 after 5 hours of Wnt3a stimulation degraded all levels of β-catenin-HaloTag-HiBiT regardless of cellular location.

Interested in more solutions for measuring targeted protein degradation?

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Protocolos

Complete Protocol

HaloPROTAC3 Technical ManualPDF (956 KB) – English

Contenido

Item	Part #	Presentación
HaloPROTAC3, 2.5mM	GA311A	1 × 20μl

SDS

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Certificado de Análisis

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Use Restrictions

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

Condiciones de Almacenaje

Patentes y Exclusiones

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 transfer or commercial use of this product 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 or its derivatives, whether or not such product or derivatives are resold for use in research. With respect to any uses outside this label license, including any commercial, 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 label license shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. Nos. 8,168,405, 8,257,939, 9,540,402 and other patents and patents pending.

Licensed from Yale University under U.S. Pat. No. 10,730,862.

Contenido

Item	Part #	Presentación
ent-HaloPROTAC3, 2.5mM	GA411A	1 × 20μl

SDS

Search for SDS

Certificado de Análisis

Search by lot number

Use Restrictions

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

Condiciones de Almacenaje

Patentes y Exclusiones

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 transfer or commercial use of this product 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 or its derivatives, whether or not such product or derivatives are resold for use in research. With respect to any uses outside this label license, including any commercial, 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 label license shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. Nos. 8,168,405, 8,257,939, 9,540,402 and other patents and patents pending.

Licensed from Yale University under U.S. Pat. No. 10,730,862.

Recursos

Menciones

Otros

Usado con frecuencia junto con

HaloTag® Fluorescent Ligands

Para el marcaje y la detección de proteínas. Cada uno incluye un grupo cloroalcano para la fijación a proteínas HaloTag®.

CS3495B06, CS3495B08, G8251, G8252, G2801, G2802, G8272, G8273, G8581, G8582, G1001, G1002, G8471, G8472, G2991, G3221, G8281, G8282, G8591, G8592

Nano-Glo® HiBiT Lytic Detection System

Método bioluminiscente para detectar la cantidad total de proteínas marcadas con HiBiT en la célula.

N3030, N3040, N3050

Small Molecule Degrader of HaloTag® Fusion Proteins

Small Molecule Degrader For Challenging Targets

How HaloPROTAC3 Promotes Target Degradation

HaloPROTAC3 and HaloTag-Edited Cells For Protein Knockout Studies