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TRIM21 pathway based Targeted Protein Degradation

Faster insights into protein function in vivo

• Avoid off-target effects by directly targeting the protein-of-interest
• Observe protein degradation in as little as 1 hour*
• Only two reagents, the Piranha System and your validated antibody
• Selective degradation of the phosphorylated or other post-translationally modified form of a protein
• Scalable for screening multiple protein targets in parallel (96- or 384-well plate assays)
• Unique system for direct targeted protein degradation for phenotypic studies
• Flexible - 3 options available:
> Electroporation-ready mRNA
> Constitutively expressed in a HEK293T cell line
> Pre-packaged lentivirus for stable Piranha expression in the cell line of your choice

*Based on published data by Clift et al., 2017, see below

How It Works
SBI’s Piranha Targeted Protein Degradation System is based on the TRIM21 pathway found in most mammalian cells1 (Figure 1). Like TRIM21, the Piranha protein binds to conserved regions of an antibody, targeting the antibody—and the bound antigen—for degradation by the proteasome.

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Read more about SBI´s Piranha Targeted Protein Degradation System:
Cell 171: 1692-1706 (2017)
A Method for the Acute and Rapid Degradation of Endogenous Proteins. Dean Clift, William A. McEwan, Larisa I. Labzin, Vera Konieczny, Binyam Mogessie, Leo C. James and Melina Schuh.


To use Piranha Electroporation-ready mRNA for targeted protein degradation, simply electroporate the Piranha mRNA and a validated antibody specific for the protein-of-interest into target cells, wait an appropriate amount of time, and then observe the phenotypic outcome.

Because the rate of protein degradation is based upon many factors, we recommend performing initial experiments to empirically determine the degradation rate of your target protein and the optimal time for phenotypic observation.


Supporting Data

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Figure 2. Piranha Electroporation-ready mRNA is pure and efficiently delivered and translated inside cells. (A) Denaturing RNA gel electrophoresis of Piranha RFP-tagged, untagged, and GFP-tagged mRNA shows clean transcripts of an appropriate size. (B) Fluorescence imaging of HEK293T cells electroporated with Piranha RFP-tagged mRNA shows robust delivery and translation of the construct.


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Figure 3. Piranha HEK293T Cells electroporated with anti-IKKá antibody show complete and near-complete ablation of IKKá after one day. A validated anti-IKKá antibody was electroporated into Piranha HEK293T cells using two different conditions (A and B), and IKKá protein levels assessed via Western blot and compared to a no-construct control. On day 1 post-electroporation, condition A shows complete ablation of IKKá protein and condition B shows almost complete ablation. By Day 2 levels of IKKá protein begin to return to pre-electroporation levels as the amount of antibody dissipates.


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Figure 4. Piranha mRNA and antibody efficiently reduce protein levels at a faster rate than corresponding siRNA. Piranha mRNA and validated anti-IêBá antibody were co-electroporated into HeLa cells (top panel) and IêBá protein levels monitored over time via Western blot. Compared to IêBá levels after introduction of IêBá siRNA (bottom panel), the Piranha System reduces IêBá protein levels faster—reduction is apparent by 8 hours (top panel), whereas for siRNA protein reduction is first apparent only after 24 hours (bottom panel).


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Figure 5. The speed of the Piranha System is also evident using a luciferase-based activity assay. MDA-MB-231 breast cancer cells were co-electroporated with Piranha mRNA and anti-IêBá antibody, and IêBá activity measured using an NFêB luciferase reporter assay—lower IêBá levels result in higher NFêB luciferase reporter activity. Compared to reduction of IêBá activity by siRNA (dark gray bars), which peaks on day 2, the Piranha System shows peak reduction of IêBá activity on day 1 (blue bars). The negative control is shown as light gray bars.

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Figure 6. The Piranha System can reduce levels of phosphorylated protein. Piranha HEK293T Cells were electroporated with either buffer only (NC), anti-phospho-AKT(S473) antibody, or an IgG control, and total AKT levels assessed via Western blot. Within 5-hrs post-electroporation, both phosphorylated and total AKT protein levels are reduced.
Related Links

pCDH-CMV-MCS-EF1a-GreenPuro Cloning and Expr. Lentivector
TransDux Max Lentivirus Transduction Enhancer
User Manual Piranha Targeted Protein Degradation System

 

 

Description Cat# Size Price    
pCDH-CMV-Piranha-EF1-GFP-T2A-Puro virus PTPD513VA-1-GVO-SBI >1x10^6 IFUs 722 € DETAILS   Add to Cart 
pCDH-EF1a-Piranha-T2A-Puro virus PTPD527VA-1-GVO-SBI >1x10^6 IFUs 722 € DETAILS   Add to Cart 
Piranha Electroporation-ready GFP-tagged mRNA PTPD520A-1-SBI 10 ug 445 € DETAILS   Add to Cart 
Piranha Electroporation-ready mRNA PTPD500A-1-SBI 10 ug 414 € DETAILS   Add to Cart 
Piranha Electroporation-ready RFP-tagged mRNA PTPD510A-1-SBI 10 ug 445 € DETAILS   Add to Cart 

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