TRAF3IP3 Antibody, HRP conjugated

What is TRAF3IP3 and why is it important in immunological research?

TRAF3 interacting protein 3 (TRAF3IP3) is an essential adapter protein containing a coiled-coil domain that plays critical roles in both innate and adaptive immunity. It is crucial for thymocyte development and regulates T-cell stability and function by recruiting the serine-threonine phosphatase catalytic subunit (PPP2CA) to the lysosome . Additionally, TRAF3IP3 is involved in antiviral signaling pathways, particularly in the RIG-I-MAVS antiviral signaling that stimulates interferon production and confers innate immunity to the host . Understanding TRAF3IP3 function is valuable for immunologists studying adaptive immune responses and antiviral mechanisms.

What is the recommended dilution range for TRAF3IP3 antibody in different applications?

Based on data from similar TRAF3IP3 antibodies, the recommended dilution ranges are:

It is important to note that optimal dilutions are sample-dependent and should be determined empirically for each experimental system to achieve the best signal-to-noise ratio .

What is the reactivity profile of TRAF3IP3 antibody (HRP conjugated)?

The TRAF3IP3 antibody (HRP conjugated) specifically targets amino acids 74-185 of human TRAF3IP3 . According to reactivity data from similar products, TRAF3IP3 antibodies can detect both human and mouse TRAF3IP3, with confirmed reactivity against human samples in published literature . When designing experiments using this antibody, it's essential to consider species compatibility and validate cross-reactivity if working with non-human models.

How can TRAF3IP3 antibody be used to study antiviral signaling pathways?

To investigate these seemingly contradictory roles, researchers can design experiments using the TRAF3IP3 antibody to:

• Track TRAF3IP3 localization during viral infection through immunofluorescence microscopy

• Monitor TRAF3IP3 protein levels in response to viral challenge via Western blotting

• Assess TRAF3IP3 interactions with key signaling proteins (MAVS, TBK1, TRAF3) through co-immunoprecipitation followed by immunoblotting with the TRAF3IP3 antibody

• Compare TRAF3IP3 expression levels between different cell types to understand tissue-specific regulation

These approaches can help elucidate the context-dependent functions of TRAF3IP3 in antiviral immunity .

What considerations should be taken when using TRAF3IP3 antibody to study proteolytic cleavage during viral infection?

Recent research has shown that TRAF3IP3 can be cleaved by the EV71 3C protease during viral infection . When investigating such post-translational modifications:

• Select appropriate experimental controls, including uninfected cells and cells expressing catalytically inactive mutants (e.g., 3C-C147S) to distinguish specific cleavage from non-specific degradation

• Consider using gradient concentrations of viral proteases to establish dose-dependent relationships

• Employ antibodies recognizing different epitopes (N-terminal vs. C-terminal) to identify cleavage fragments

• Design time-course experiments to track the kinetics of TRAF3IP3 cleavage during infection

The detection of cleavage products requires careful optimization of gel electrophoresis conditions to resolve fragments of similar molecular weights. Western blotting using the HRP-conjugated TRAF3IP3 antibody can provide valuable insights into how viruses manipulate host antiviral mechanisms .

How can researchers address potential cross-reactivity issues with TRAF3IP3 antibody in multiprotein complex studies?

When studying TRAF3IP3 interactions within multiprotein complexes:

• Validate antibody specificity using knockout/knockdown controls (TRAF3IP3-deficient cells) to confirm signal specificity

• Perform blocking peptide experiments with the immunizing peptide (amino acids 74-185) to confirm epitope specificity

• Consider potential confounding factors from proteins with similar epitopes or molecular weights

• Use orthogonal detection methods to confirm co-immunoprecipitation results

These validation steps are particularly important when studying TRAF3IP3 interactions with its known binding partners such as TRAF3, MAVS, and TBK1 in antiviral signaling complexes .

What is the optimal protocol for detecting TRAF3IP3 in Western blotting applications?

When performing Western blotting for TRAF3IP3 detection:

• Prepare cell lysates in RIPA buffer with protease inhibitors

• Separate proteins on 10-12% SDS-PAGE gels (TRAF3IP3 has reported molecular weights of 41 kDa, 47 kDa, and 64 kDa)

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk or BSA in TBST for 1 hour

• Incubate with diluted TRAF3IP3-HRP antibody (1:500-1:3000) for 2 hours at room temperature or overnight at 4°C

• Wash thoroughly with TBST (3-5 times, 5 minutes each)

• Develop using ECL substrate directly (no secondary antibody needed due to HRP conjugation)

Note that TRAF3IP3 can appear at different molecular weights (observed at 64 kDa, 47 kDa, and 41 kDa) , so proper positive controls are essential for accurate band identification.

What are the common technical challenges when using TRAF3IP3 antibody in immunohistochemistry?

Researchers using TRAF3IP3 antibodies for IHC applications should consider these challenges:

• Antigen retrieval method selection: Data suggests that TE buffer pH 9.0 is recommended for optimal antigen retrieval, although citrate buffer pH 6.0 may be used as an alternative

• Tissue-specific optimization: Positive detection has been confirmed in human colon cancer tissue and human tonsillitis tissue

• Signal specificity: Include appropriate positive controls (Jurkat cells, human colon cancer tissue, mouse thymus tissue) and negative controls (tissues known not to express TRAF3IP3)

• Background reduction: Optimize blocking conditions and antibody dilutions to minimize non-specific staining

When troubleshooting weak or absent signals, consider increasing antibody concentration, extending incubation times, or testing alternative antigen retrieval methods.

How can TRAF3IP3 antibody be validated for studying virus-host interactions?

To validate TRAF3IP3 antibody performance in virus-host interaction studies:

• Compare TRAF3IP3 detection in mock-infected versus virus-infected cells

• Use TRAF3IP3 knockout/knockdown cells as negative controls

• Verify antibody specificity against recombinant TRAF3IP3 protein

• Perform parallel experiments with multiple antibodies targeting different TRAF3IP3 epitopes

These validation steps are particularly important when studying TRAF3IP3 cleavage by viral proteases like EV71 3C or changes in TRAF3IP3 localization during infection.

How should researchers interpret variations in TRAF3IP3 molecular weight across different experimental systems?

TRAF3IP3 can be detected at multiple molecular weights (64 kDa, 47 kDa, and 41 kDa) , which may reflect:

• Alternative splicing variants (the calculated molecular weights are 41 kDa for a 353aa variant and 64 kDa for a 551aa variant)

• Post-translational modifications such as phosphorylation or ubiquitination

• Proteolytic processing during sample preparation or in biological contexts

• Cell type-specific expression patterns

When encountering unexpected molecular weight variations, researchers should consider:

• Running molecular weight standards alongside samples

• Including known positive controls from validated cell lines (e.g., Jurkat cells)

• Using epitope-mapped antibodies to determine which protein region is being detected

• Performing additional experiments such as mass spectrometry to confirm protein identity

How can researchers reconcile contradictory findings regarding TRAF3IP3's role in antiviral immunity?

The literature presents seemingly contradictory roles for TRAF3IP3 in antiviral immunity:

• Negative regulatory role: TRAF3IP3 has been shown to suppress cytosolic RNA-induced IFN-I pathway by inhibiting IRF3 phosphorylation and nuclear translocation

• Positive regulatory role: TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for effective antiviral innate immune responses

To address these contradictions, researchers should:

• Consider cell type-specific effects (TRAF3IP3 expression varies across different cell types)

• Examine the timing of responses (early vs. late responses in infection)

• Evaluate the viral stimulus used (different viruses may trigger distinct pathways)

• Assess experimental conditions (overexpression vs. knockdown approaches)

• Design experiments that directly compare TRAF3IP3 function across multiple cell types and viral challenges

A comprehensive experimental approach combining both loss-of-function and gain-of-function studies can help clarify these context-dependent roles.

What considerations should be made when designing co-immunoprecipitation experiments with TRAF3IP3 antibody?

When planning co-immunoprecipitation (Co-IP) experiments to study TRAF3IP3 protein interactions:

• Antibody orientation: Consider whether the HRP conjugation might interfere with epitope binding during immunoprecipitation; for Co-IP, unconjugated antibodies may be preferable

• Buffer conditions: Optimize lysis buffer composition to preserve protein-protein interactions (mild detergents like NP-40 or Triton X-100 are preferable to RIPA buffer)

• Control selection: Include isotype controls and reciprocal Co-IPs to confirm specific interactions

• Domain mapping: When examining interactions with proteins like TBK1, consider using deletion constructs to map interaction domains as demonstrated in the literature

What emerging applications could benefit from TRAF3IP3 antibody-based detection methods?

Future research applications for TRAF3IP3 antibody may include:

• Single-cell analysis: Combining TRAF3IP3 antibody with flow cytometry or mass cytometry to examine expression at the single-cell level

• Proximity ligation assays: Using TRAF3IP3 antibody in combination with antibodies against interaction partners to visualize protein complexes in situ

• ChIP-seq applications: Investigating potential chromatin interactions if TRAF3IP3 has nuclear functions

• Therapeutic target validation: Evaluating TRAF3IP3 as a potential target in antiviral therapies or immune modulation

These applications could provide deeper insights into TRAF3IP3's multifaceted roles in immunity and viral pathogenesis.

How might TRAF3IP3 antibodies contribute to understanding emerging viral pathogen responses?

TRAF3IP3 antibodies could be valuable tools for studying host responses to emerging viral pathogens by:

• Tracking changes in TRAF3IP3 expression, localization, or post-translational modifications during infection

• Identifying viral immune evasion strategies that target TRAF3IP3 function

• Examining how TRAF3IP3-dependent signaling contributes to pathogen-specific responses

• Screening for viral proteins that interact with or modify TRAF3IP3

Given that TRAF3IP3 is cleaved by EV71 3C protease , similar mechanisms might exist for other viruses, representing an important area for future investigation.