USP13 Antibody

What is USP13 and what biological functions does it serve?

USP13 (ubiquitin-specific peptidase 13, also known as ISOT3) is an 863 amino acid protein belonging to the deubiquitinating enzyme (DUB) family. It plays a crucial role in the ubiquitin-proteasome system, which is essential for maintaining cellular homeostasis by regulating protein degradation and turnover. USP13 is primarily located in both the cytoplasm and nucleus, where it catalyzes the removal of ubiquitin from substrate proteins, thereby influencing various cellular functions including signal transduction and transcriptional regulation . With a unique structure featuring one UBP-type zinc finger and two UBA domains, USP13 is especially highly expressed in testicular and ovarian tissues, underscoring its significance in reproductive biology . Recent studies have identified its important role in the negative regulation of antiviral immunity through deubiquitination of STING, a critical component of innate immune responses against viral infections .

What types of USP13 antibodies are available for research applications?

Several monoclonal antibodies targeting USP13 are available for research purposes. These include:

• USP13 Antibody (D-11): A mouse monoclonal IgG1 antibody that detects USP13 in mouse, rat, and human samples .

• USP13 Antibody (B-9): A mouse monoclonal IgG2b kappa light chain antibody that recognizes USP13 protein across mouse, rat, and human species .

• Anti-USP13 antibody [EPR4348]: A rabbit recombinant antibody option for detecting USP13 .

These antibodies are available in various forms, including non-conjugated versions and conjugated forms with agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates to accommodate different experimental needs and detection methods .

What experimental applications can USP13 antibodies be used for?

USP13 antibodies have been validated for multiple experimental applications critical to molecular and cellular biology research. The primary applications include:

• Western Blotting (WB): For detecting USP13 protein in cell or tissue lysates, allowing quantification and size determination.

• Immunoprecipitation (IP): For isolating USP13 and its binding partners from complex protein mixtures.

• Immunofluorescence (IF): For visualizing the subcellular localization and distribution patterns of USP13 within cells.

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of USP13 in solution .

These diverse applications make USP13 antibodies versatile tools for investigating the protein's expression, interactions, and functions in various experimental contexts.

How should I design experiments to study USP13's deubiquitinating activity?

When investigating USP13's deubiquitinating activity, consider implementing a multi-faceted experimental approach:

• In vitro deubiquitination assays: Use purified recombinant USP13 and model substrates such as glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase. Include both wild-type USP13 and catalytically inactive mutant (C345S) as controls to confirm specificity of the deubiquitinating activity .

• Cell-based ubiquitination detection: Overexpress USP13 in cell lines (293T cells work well) alongside substrate proteins of interest. Use immunoprecipitation with USP13 antibodies followed by western blotting with anti-ubiquitin antibodies to detect changes in substrate ubiquitination status .

• Proteomic approaches: Consider differential proteomics using techniques like 2D-DIGE to identify cellular proteins whose expression is significantly altered following USP13 overexpression or knockdown. Compare results between wild-type USP13 and the C345S catalytically inactive mutant to distinguish between enzymatic and scaffolding functions .

• Controls: Always include the catalytically inactive USP13 C345S mutant (cysteine 345 mutated to serine) in parallel experiments to distinguish between ubiquitin-dependent and independent functions .

This comprehensive approach will provide robust evidence for USP13's deubiquitinating function and potential substrates.

What are the optimal conditions for using USP13 antibodies in Western blotting?

For optimal Western blotting results with USP13 antibodies, follow these methodological guidelines:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. For detecting USP13 (approximately 97 kDa), prepare 10-12% SDS-PAGE gels.

• Protein loading and transfer: Load 20-50 μg of total protein per lane. Transfer to PVDF membranes (preferred over nitrocellulose for this protein) at 100V for 90 minutes using standard transfer buffer with 10% methanol.

• Blocking and antibody incubation: Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature. Dilute primary USP13 antibodies (such as D-11 or B-9) at 1:1000 in 5% BSA in TBST and incubate overnight at 4°C .

• Detection and visualization: Use appropriate secondary antibodies conjugated to HRP at 1:5000 dilution for 1 hour at room temperature. For enhanced sensitivity, consider using m-IgG Fc BP-HRP bundle options available with these antibodies .

• Controls: Include positive control samples from tissues known to express high levels of USP13 (testicular or ovarian tissues) alongside experimental samples .

This protocol has been validated with commercially available USP13 antibodies and yields specific detection of the protein across multiple species including human, mouse, and rat samples.

How can I visualize USP13 subcellular localization using immunofluorescence?

For precise visualization of USP13 subcellular localization using immunofluorescence:

• Cell preparation: Culture cells on glass coverslips to 70-80% confluence. Fix with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes.

• Antibody selection: Choose fluorophore-conjugated USP13 antibodies (such as FITC or Alexa Fluor® conjugates) for direct detection, or use unconjugated primary antibodies followed by fluorophore-conjugated secondary antibodies .

• Immunostaining protocol:

  • Block with 5% normal serum in PBS for 1 hour

  • Incubate with primary USP13 antibody (1:100-1:200 dilution) overnight at 4°C

  • Wash thoroughly with PBS (3×5 minutes)

  • If using unconjugated primary antibody, incubate with appropriate fluorescent secondary antibody (1:500) for 1 hour at room temperature

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

• Co-localization studies: For investigating USP13's interaction with other proteins, perform double immunofluorescence staining with antibodies against potential interacting partners like STING . Use different fluorophores (e.g., red for partner protein, green for USP13) to visualize co-localization.

• Imaging parameters: Capture images using confocal microscopy with appropriate laser settings for the selected fluorophores. For USP13, expect to observe both cytoplasmic and nuclear localization patterns .

This methodology enables detailed visualization of USP13's distribution within cells and its potential co-localization with interacting proteins.

How does USP13 regulation impact antiviral immune responses?

USP13 serves as a negative regulator of innate antiviral immunity through its deubiquitinating activity on STING (stimulator of interferon genes), a critical component of cellular defense against DNA viruses. The relationship between USP13 and antiviral responses involves several mechanistic aspects:

• STING regulation: USP13 directly interacts with STING and catalyzes the removal of polyubiquitin chains, which are required for STING activation and downstream signaling. This deubiquitination prevents the recruitment of TANK-binding kinase 1 (TBK1) to the signaling complex .

• Impact on signaling pathways: USP13 knockdown or knockout potentiates the activation of IRF3 and NF-κB transcription factors following HSV-1 infection or transfection with DNA ligands, leading to enhanced expression of downstream antiviral genes .

• Viral replication control: USP13 deficiency results in impaired replication of HSV-1 in experimental models. Consistent with this observation, USP13-deficient mice exhibit greater resistance to lethal HSV-1 infection compared to wild-type littermates .

• Experimental approach to studying this relationship:

  • Generate USP13 knockout cell lines using CRISPR/Cas9

  • Challenge with DNA viruses (e.g., HSV-1) or cytosolic DNA mimics

  • Measure activation of IRF3/NF-κB (by phosphorylation status)

  • Quantify interferon and pro-inflammatory cytokine production

  • Assess viral replication through plaque assays or qPCR for viral genes

This research area highlights USP13 as a potential therapeutic target for enhancing antiviral immunity, particularly against DNA viruses that trigger the STING-dependent pathway.

What techniques can be used to identify novel USP13 substrates in different cellular contexts?

Identifying novel USP13 substrates requires a multi-dimensional approach combining biochemical, proteomic, and genetic techniques:

• Comparative proteomics:

  • Implement stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry to compare ubiquitinome profiles between USP13 wild-type and knockout conditions

  • Use 2D-DIGE to detect cellular proteins whose expression is significantly altered following USP13 overexpression or knockdown

  • Compare results between wild-type USP13 and the C345S catalytically inactive mutant to distinguish true substrates from non-specific interactions

• Affinity purification-based approaches:

  • Perform immunoprecipitation with USP13 antibodies followed by mass spectrometry to identify interacting proteins

  • Use tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins, then compare profiles between USP13-deficient and control cells

  • Implement BioID or proximity labeling approaches with USP13 as the bait to identify proteins in close proximity

• Genetic screens:

  • Deploy CRISPR-Cas9 screens to identify genes that synthetically interact with USP13

  • Use global protein stability profiling in USP13-deficient backgrounds to identify proteins whose stability is regulated by USP13

• Validation of putative substrates:

  • Confirm direct deubiquitination using in vitro deubiquitination assays with recombinant USP13 and immunopurified substrates

  • Perform ubiquitination assays in cells with USP13 overexpression, knockdown, or the C345S mutant

  • Analyze ubiquitination chain topology to determine the type of chains (K48, K63, etc.) targeted by USP13

These approaches have successfully identified several USP13 substrates in different cellular contexts, including those involved in DNA damage responses, metabolic regulation, and immune signaling pathways.

How can USP13 antibodies be used to investigate the role of USP13 in disease models?

USP13 antibodies are instrumental in investigating the role of this deubiquitinase in various disease models, particularly in cancer, neurodegenerative disorders, and viral infections:

• Cancer research applications:

  • Use immunohistochemistry with USP13 antibodies on tissue microarrays to assess correlation between USP13 expression and clinical outcomes

  • Perform western blotting to compare USP13 protein levels across patient samples and matched normal tissues

  • Investigate USP13 interaction with known cancer-related proteins through co-immunoprecipitation followed by immunoblotting

• Neurodegenerative disease models:

  • Employ immunofluorescence to examine USP13 colocalization with protein aggregates in models of neurodegenerative diseases

  • Use USP13 antibodies to assess changes in expression or localization during disease progression

  • Implement proximity ligation assays to detect interactions between USP13 and disease-relevant proteins in situ

• Viral infection studies:

  • Monitor changes in USP13 expression and localization during viral infection using western blotting and immunofluorescence

  • Investigate USP13's interaction with viral proteins through co-immunoprecipitation

  • Assess the role of USP13 in viral replication by combining antibody-based detection with viral load measurements

• Methodological considerations:

  • For tissue sections, optimize antigen retrieval methods (citrate buffer, pH 6.0 at 95°C for 20 minutes works well for most USP13 antibodies)

  • Validate antibody specificity using USP13 knockout tissues as negative controls

  • Consider using multiple antibody clones (D-11, B-9) targeting different epitopes to confirm findings

These approaches enable comprehensive analysis of USP13's involvement in pathological processes and potential as a therapeutic target.

What are common challenges when working with USP13 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with USP13 antibodies. Here are common issues and methodological solutions:

• Non-specific bands in Western blotting:

  • Increase blocking stringency by using 5% BSA instead of milk

  • Optimize primary antibody dilution (start with 1:1000 and adjust as needed)

  • Include a peptide competition assay using USP13 (D-11) Neutralizing Peptide to confirm band specificity

  • Try alternative USP13 antibody clones (switch between D-11 and B-9) that may offer better specificity for your particular sample type

• Weak signal detection:

  • Increase protein loading (50-75 μg per lane)

  • Extend primary antibody incubation to overnight at 4°C

  • Consider using signal enhancement systems such as the m-IgG Fc BP-HRP Bundle option available with commercial USP13 antibodies

  • Implement epitope retrieval techniques for fixed samples

• Inconsistent immunoprecipitation results:

  • Pre-clear lysates with protein A/G beads before immunoprecipitation

  • Consider crosslinking antibodies to beads to prevent antibody contamination in eluted samples

  • Use agarose-conjugated USP13 antibody formulations for more efficient pulldown

  • Optimize lysis conditions to preserve protein interactions (milder detergents like NP-40)

• Background in immunofluorescence:

  • Implement additional washing steps (5× instead of 3×)

  • Use centrifugation-based washing for suspension cells

  • Include 0.1% Tween-20 in wash buffers to reduce non-specific binding

  • Optimize fixation methods (compare paraformaldehyde vs. methanol fixation)

These methodological adjustments can significantly improve experimental outcomes when working with USP13 antibodies across different applications and sample types.

How should researchers interpret and validate USP13 antibody results across different experimental systems?

Proper interpretation and validation of USP13 antibody results require rigorous controls and comparative analysis across experimental systems:

• Cross-validation with multiple antibodies:

  • Compare results obtained with different USP13 antibody clones (D-11, B-9, EPR4348) targeting distinct epitopes

  • If discrepancies arise, prioritize results that are consistent across multiple antibodies

• Genetic validation approaches:

  • Include USP13 knockout or knockdown samples as negative controls

  • Perform rescue experiments by reintroducing USP13 into knockout systems

  • Use the catalytically inactive USP13 C345S mutant to distinguish between enzymatic and scaffolding functions

• Cross-species validation:

  • Confirm findings across multiple species (human, mouse, rat) when possible, as most USP13 antibodies recognize the protein across these species

  • Be aware of potential species-specific differences in USP13 function or regulation

• Cross-technique validation:

  • Confirm key findings using complementary techniques (e.g., if detecting increased expression by Western blot, validate with qPCR)

  • For interaction studies, confirm co-immunoprecipitation results with proximity ligation assays or FRET

  • Validate subcellular localization using both immunofluorescence and subcellular fractionation followed by Western blotting

• Quantitative analysis approaches:

  • Implement densitometric analysis for Western blots with appropriate normalization to loading controls

  • For immunofluorescence, use quantitative image analysis measuring colocalization coefficients (Pearson's or Manders')

  • Apply statistical tests appropriate for the experimental design and data distribution

This systematic validation approach ensures robust and reproducible results when using USP13 antibodies across different experimental contexts.

What quantitative methods can be used to accurately measure USP13 expression and activity in experimental samples?

Accurately quantifying USP13 expression and activity requires a combination of protein-level, mRNA-level, and functional assays:

• Protein expression quantification:

  • Western blotting with USP13 antibodies followed by densitometric analysis, normalized to appropriate loading controls (β-actin, GAPDH)

  • ELISA using commercially available USP13 antibodies for absolute quantification in solution samples

  • Flow cytometry for single-cell analysis of USP13 expression using fluorophore-conjugated USP13 antibodies

  • Mass spectrometry-based targeted proteomics using selected reaction monitoring (SRM) for absolute quantification

• mRNA expression analysis:

  • Quantitative RT-PCR for USP13 mRNA levels, normalized to stable reference genes

  • RNA-seq for genome-wide expression analysis, including USP13 transcript variants

  • Northern blotting for confirmation of transcript size and abundance

• USP13 deubiquitinating activity measurement:

  • In vitro deubiquitination assays using fluorogenic ubiquitin substrates

  • Cell-based reporter systems with ubiquitin-luciferase fusion constructs

  • Targeted analysis of known substrate ubiquitination status (e.g., STING) using ubiquitin antibodies after USP13 modulation

  • DUB activity assays using ubiquitin-β-galactosidase as a model substrate, with subsequent colorimetric or fluorometric detection

• Data normalization and analysis:

  • For tissue samples, normalize USP13 levels to tissue-specific reference proteins

  • Account for cell-type specific expression patterns when analyzing heterogeneous samples

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • Present data with clear indication of biological and technical replicates

This multi-faceted approach provides comprehensive quantitative assessment of both USP13 expression and functional activity in experimental systems.

How might USP13 antibodies be utilized in developing new therapeutic strategies?

USP13 antibodies have significant potential in therapeutic development strategies, particularly in the following areas:

• Target validation for drug development:

  • Use USP13 antibodies in immunohistochemistry to stratify patient samples based on USP13 expression levels

  • Employ antibody-based screening assays to identify small molecule inhibitors that disrupt USP13's interaction with specific substrates

  • Implement proximity-based assays with USP13 antibodies to confirm engagement of candidate drugs with USP13 in cellular contexts

• Immunotherapy approaches:

  • Develop antibody-drug conjugates targeting USP13 in diseases where it is overexpressed

  • Explore intrabody approaches where USP13 antibodies are expressed intracellularly to modulate its function

  • Investigate CAR-T cell approaches for conditions with aberrant USP13 expression on cell surfaces

• Diagnostic and companion diagnostic applications:

  • Use USP13 antibodies to develop diagnostic assays for diseases with altered USP13 expression

  • Create companion diagnostics to identify patients likely to respond to USP13-targeting therapies

  • Develop prognostic assays based on USP13 expression patterns in disease tissues

• Enhancing antiviral immunity:

  • Target USP13 inhibition to enhance STING-mediated antiviral responses, particularly against DNA viruses like HSV-1

  • Develop checkpoint inhibitor-like approaches that block USP13's interaction with STING to boost innate immunity

  • Use USP13 antibodies to monitor treatment efficacy in these approaches

These therapeutic strategies leverage the fundamental biological insights into USP13 function gained through research applications of USP13 antibodies, demonstrating the translational potential of basic research tools.

What are the latest methodological advances in studying USP13's role in cellular signaling networks?

Recent methodological advances have significantly enhanced our ability to study USP13's complex roles in cellular signaling networks:

• Proximity-dependent labeling techniques:

  • BioID and TurboID approaches with USP13 as the bait protein to identify the USP13 proximitome

  • APEX2-based proximity labeling for temporal control of labeling, allowing investigation of dynamic USP13 interactions

  • Split-BioID systems to study context-specific USP13 interactions within particular cellular compartments

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) with USP13 antibodies to visualize nanoscale distribution patterns

  • Live-cell imaging using fluorescently tagged USP13 combined with substrate proteins to monitor deubiquitination dynamics

  • Correlative light and electron microscopy (CLEM) to examine USP13's ultrastructural localization

• Systems biology integration:

  • Network analysis incorporating USP13 interactions, substrates, and expression patterns

  • Computational modeling of USP13's impact on ubiquitin signaling networks

  • Multi-omics integration connecting USP13-dependent changes across transcriptome, proteome, and ubiquitinome

• CRISPR-based technologies:

  • CRISPRi/CRISPRa for tunable modulation of USP13 expression

  • Base editing to introduce specific point mutations in USP13 (such as the C345S catalytic mutant) at endogenous loci

  • CRISPR screens to identify synthetic interactions with USP13 in different cellular contexts

• Protein engineering approaches:

  • Development of USP13 activity-based probes that covalently bind to active enzyme

  • Engineered ubiquitin variants that specifically inhibit or enhance USP13 activity

  • Optogenetic control of USP13 localization or activity to study temporal aspects of signaling

These methodological advances provide unprecedented insights into USP13's functions within complex cellular signaling networks and open new avenues for therapeutic targeting.

How should researchers select the optimal USP13 antibody for their specific experimental needs?

Selecting the most appropriate USP13 antibody requires careful consideration of several technical parameters:

• Experimental application matching:

  • For Western blotting: Both USP13 Antibody (D-11) and (B-9) perform well, with both detecting the expected ~97 kDa band

  • For immunoprecipitation: Consider using USP13 Antibody (B-9) AC, which is pre-conjugated to agarose for efficient pulldown

  • For immunofluorescence: Fluorophore-conjugated options like USP13 Antibody (B-9) FITC provide direct detection without secondary antibodies

  • For multiplex applications: Select antibodies raised in different host species (mouse vs. rabbit) to avoid cross-reactivity

• Species reactivity requirements:

  • If working across species, select antibodies validated for cross-reactivity (both D-11 and B-9 detect human, mouse, and rat USP13)

  • For species-specific studies, verify epitope conservation in the target species

• Epitope considerations:

  • Choose antibodies targeting different epitopes for validation purposes

  • Consider epitope location relative to functional domains for studies of protein interactions

  • For detecting specific post-translational modifications, select antibodies that do not have epitopes overlapping with modification sites

• Technical specifications:

  • Antibody format: Consider monoclonal (D-11, B-9) for consistency across experiments or recombinant (EPR4348) for renewable supply

  • Conjugations: Select based on detection method requirements (HRP for chemiluminescence, fluorophores for microscopy)

  • Concentration: Standard concentrations of 200 μg/ml are suitable for most applications

• Validation data assessment:

  • Review available validation data including Western blot images, immunofluorescence patterns

  • Check for validation in knockout/knockdown systems

  • Consider published literature where specific antibody clones have been successfully used

This systematic selection process ensures optimal antibody performance for specific experimental contexts while minimizing technical artifacts.

What considerations are important when using USP13 antibodies for in vivo studies or primary tissue analysis?

When extending USP13 antibody applications to in vivo studies or primary tissue analysis, researchers should consider these critical methodological factors:

• Tissue fixation and processing optimization:

  • Compare multiple fixation methods (4% PFA, methanol, acetone) to identify optimal preservation of USP13 epitopes

  • For formalin-fixed paraffin-embedded (FFPE) tissues, implement antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • For frozen sections, ensure proper tissue freezing techniques to maintain cellular architecture

• Background reduction strategies:

  • Include blocking steps with species-specific serum matching the secondary antibody host

  • Implement avidin/biotin blocking for tissues with endogenous biotin

  • Consider autofluorescence quenching methods for tissues with high autofluorescence (e.g., liver, brain)

• Controls for tissue studies:

  • Include isotype controls at the same concentration as primary antibodies

  • Use tissues from USP13 knockout animals as negative controls

  • Implement peptide competition controls using USP13 neutralizing peptides

• Primary cell considerations:

  • Adjust permeabilization conditions for primary cells, which may differ from cell lines

  • Consider cell type-specific expression patterns when interpreting results

  • Account for culture-induced changes in USP13 expression compared to in vivo conditions

• In vivo imaging considerations:

  • For in vivo imaging applications, consider fluorophores with emission in the near-infrared range to maximize tissue penetration

  • Evaluate biodistribution and clearance characteristics of antibody conjugates

  • Implement methods to reduce non-specific binding in vivo (PEGylation, Fc modifications)

• Quantification approaches:

  • Develop scoring systems for immunohistochemistry based on staining intensity and percentage of positive cells

  • Use digital pathology tools for unbiased quantification of USP13 expression in tissue sections

  • Implement multi-parameter analysis to correlate USP13 expression with other tissue markers

These methodological considerations ensure robust and reproducible results when extending USP13 antibody applications from cell culture to more complex biological systems.