TRAF3IP3 Antibody, Biotin conjugated

What is TRAF3IP3 and what are its key functions in cellular immunity?

TRAF3IP3, also known as T3JAM or DJ434O14.3, is a 64kDa protein that plays critical roles in multiple immune signaling pathways. Research demonstrates that TRAF3IP3 functions primarily in:

• Antiviral immunity: TRAF3IP3 mediates the recruitment of TRAF3 to MAVS (mitochondrial antiviral signaling protein) during RNA virus infection, facilitating TBK1-IRF3 activation and type I interferon production

• B cell development: TRAF3IP3 is essential for development of common lymphoid progenitors (CLPs) and marginal zone (MZ) B cells in the spleen

• Autophagy regulation: TRAF3IP3 promotes autophagy through an ATG16L1-binding motif, contributing to MZ B cell survival

Knockout studies have revealed that TRAF3IP3-deficient mice exhibit significantly reduced CLPs, impaired B cell development in bone marrow, absence of MZ B cells in the spleen, reduced natural antibody levels, and compromised T cell-independent type II immune responses .

What specific epitope does the Biotin-conjugated TRAF3IP3 antibody recognize?

The Biotin-conjugated TRAF3IP3 antibody (ABIN7172345) specifically recognizes amino acids 74-185 of the human TRAF3IP3 protein . This internal region recognition contributes to the antibody's specificity for detecting endogenous levels of total TRAF3IP3 protein. The antibody was generated using a recombinant fragment of human TRAF3-interacting protein corresponding to this amino acid sequence .

For comparison, other commercially available TRAF3IP3 antibodies may target different epitopes, such as the internal region between amino acids 231-330 or regions derived from internal residues of the human protein . This epitope variability should be considered when selecting antibodies for specific experimental applications.

What experimental applications is the Biotin-conjugated TRAF3IP3 antibody validated for?

The Biotin-conjugated TRAF3IP3 antibody has been validated for multiple research applications, each with specific advantages:

The biotin conjugation provides enhanced detection sensitivity through the strong biotin-streptavidin interaction, making this antibody particularly valuable for applications requiring signal amplification. The antibody has been protein G purified to >95% purity, ensuring high specificity for research applications .

How can I optimize Western blotting protocols when using TRAF3IP3 antibody?

For optimal Western blot results with the Biotin-conjugated TRAF3IP3 antibody, implement the following research-validated protocol:

Sample preparation:

• Extract proteins using RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if phosphorylation status is relevant

• Maintain samples at 4°C throughout processing to prevent degradation

Electrophoresis and transfer conditions:

• Load 25-50μg of total protein per lane

• Use 10% SDS-PAGE gels for optimal resolution of the 64kDa TRAF3IP3 protein

• Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

Antibody incubation:

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Dilute the Biotin-conjugated TRAF3IP3 antibody at 1:500-1:1000 in TBST with 1% BSA

• Incubate overnight at 4°C with gentle agitation

• Wash extensively (4 × 10 minutes with TBST)

• Incubate with streptavidin-HRP (1:5000) for 1 hour at room temperature

• Develop using ECL substrate with appropriate exposure time

Experimental validation has shown that extending washing steps significantly improves signal-to-noise ratio when detecting endogenous TRAF3IP3. For virus infection studies, comparing samples from different time points post-infection can reveal changes in TRAF3IP3 expression and modification status .

What is the recommended protocol for immunofluorescence staining with TRAF3IP3 antibody?

For effective immunofluorescence studies using the Biotin-conjugated TRAF3IP3 antibody, particularly when investigating its subcellular localization during viral infection, follow this detailed protocol:

Cell preparation:

• Culture cells on glass coverslips to 60-70% confluence

• For viral infection studies, infect cells with the virus of interest (e.g., Sendai virus) for 10-24 hours

Staining procedure:

• For mitochondrial colocalization studies, stain live cells with 150nM MitoTracker Red CMXRos for 15 minutes at room temperature

• Wash cells with pre-warmed PBS (3 times)

• Fix cells with 4% formaldehyde for 15 minutes at room temperature

• Permeabilize with 0.5% Triton X-100 for 15 minutes

• Block with 3% BSA in PBS for 1 hour

• Incubate with Biotin-conjugated TRAF3IP3 antibody (1:200-1:500 dilution) overnight at 4°C

• Wash with PBS (3 × 5 minutes)

• Incubate with streptavidin-conjugated fluorophore (e.g., streptavidin-FITC) for 1 hour at room temperature

• For co-localization studies, include appropriate organelle markers such as anti-calnexin (ER) or anti-GM130 (Golgi)

• Counterstain nuclei with DAPI

• Mount slides with anti-fade mounting medium

• Image using confocal microscopy

Research using this approach has demonstrated that upon viral infection, TRAF3IP3 significantly accumulates on mitochondria, which can be visualized as colocalization between the TRAF3IP3 signal and MitoTracker staining .

How can I use TRAF3IP3 antibody to study antiviral signaling pathways?

The Biotin-conjugated TRAF3IP3 antibody can be effectively employed to investigate the RIG-I-MAVS antiviral signaling pathway through several methodological approaches:

Protein-protein interaction studies:

• Co-immunoprecipitation: Use the biotin-conjugated antibody to pull down TRAF3IP3 and probe for interaction partners like MAVS and TRAF3

• Analyze how these interactions change during viral infection time course (0h, 6h, 12h, 24h)

Subcellular localization analysis:

• Track TRAF3IP3 relocalization to mitochondria during viral infection using immunofluorescence

• Quantify colocalization with MAVS at the mitochondrial membrane

Functional signaling assays:

• Combine with IRF3 dimerization assays to correlate TRAF3IP3 recruitment with downstream signaling activation

• Use in conjunction with interferon promoter reporter assays to link TRAF3IP3 activity to interferon production

Research has established that TRAF3IP3 functions specifically in the TBK1-IRF3 activation branch of RIG-I signaling but not the IKK-NF-κB branch. Upon viral infection, TRAF3IP3 accumulates on mitochondria and facilitates TRAF3 recruitment to MAVS, which is essential for TBK1-IRF3 activation and type I interferon production .

Experimental evidence shows that TRAF3IP3 deficiency cripples RIG-I- and MAVS-triggered IFNB production, while TBK1- or IRF3-triggered IFNB induction remains unaffected, confirming TRAF3IP3's positioning in the signaling cascade between MAVS and TBK1 .

How does TRAF3IP3 localization change during viral infection and what methodology best captures this?

TRAF3IP3 undergoes significant relocalization during viral infection, providing a dynamic marker for antiviral response activation. This relocalization can be methodically analyzed through:

Subcellular fractionation:

• Separate cellular compartments (cytosol, mitochondria, ER, nucleus) through differential centrifugation

• Process infected and uninfected cells to obtain P5 (mitochondria-enriched) and S5 fractions

• Analyze TRAF3IP3 distribution by Western blotting of fractions using the biotin-conjugated antibody

• Quantify the mitochondrial/cytosolic ratio of TRAF3IP3 at different time points post-infection

Live-cell imaging:

• Express fluorescently-tagged TRAF3IP3 and use the antibody to validate that the tagged protein behaves similarly to endogenous protein

• Perform time-lapse imaging during viral infection to track dynamic relocalization

Immunofluorescence microscopy:

• Fix cells at defined time points after infection (0h, 6h, 12h, 24h)

• Co-stain for TRAF3IP3 and organelle markers (MitoTracker, anti-calnexin, anti-GM130)

• Quantify colocalization coefficients between TRAF3IP3 and each organelle marker

Research has demonstrated that in uninfected cells, TRAF3IP3 shows diffuse cytoplasmic distribution, but upon RNA virus infection (e.g., Sendai virus), it significantly accumulates on mitochondria where it colocalizes with MAVS . This mitochondrial accumulation peaks approximately 12 hours post-infection and correlates with the timing of type I interferon induction.

What is the role of TRAF3IP3 in autophagy and how can I study it?

TRAF3IP3 promotes autophagy through an ATG16L1-binding motif, which is critical for marginal zone B cell survival. To investigate this function using the Biotin-conjugated TRAF3IP3 antibody, researchers can implement these methodological approaches:

Autophagosome formation assay:

• Transfect cells with GFP-LC3 (autophagosome marker)

• Stimulate or inhibit autophagy with rapamycin or bafilomycin A1, respectively

• Immunostain for TRAF3IP3 using the biotin-conjugated antibody

• Analyze colocalization between TRAF3IP3 and LC3-positive autophagosomes

ATG16L1 interaction analysis:

• Perform co-immunoprecipitation using the biotin-conjugated TRAF3IP3 antibody

• Blot for ATG16L1 to confirm the interaction

• Create deletion mutants to map the interaction domains

Autophagic flux measurement:

• Compare LC3-I to LC3-II conversion and p62 degradation in wild-type versus TRAF3IP3-deficient cells

• Use the TRAF3IP3 antibody to confirm knockout/knockdown efficiency

Research has revealed that TRAF3IP3-knockout mice exhibit diminished autophagy in marginal zone B cells, leading to increased apoptosis and ultimately the absence of this B cell population . This suggests that TRAF3IP3's autophagy-promoting function is essential for MZ B cell survival and their contribution to T cell-independent type II immune responses.

How can I design co-immunoprecipitation experiments using Biotin-conjugated TRAF3IP3 antibody?

The biotin conjugation of the TRAF3IP3 antibody provides distinct advantages for co-immunoprecipitation experiments. To optimize these experiments:

Standard co-IP protocol:

• Prepare cell lysates in mild lysis buffer (1% NP-40, 150mM NaCl, 20mM Tris-HCl pH 7.4, 1mM EDTA, protease inhibitors)

• Pre-clear lysates with protein G-agarose for 1 hour at 4°C

• Incubate pre-cleared lysates with Biotin-conjugated TRAF3IP3 antibody (2-5μg per 1mg protein) overnight at 4°C

• Add streptavidin-coated magnetic beads and incubate for 2 hours at 4°C

• Wash extensively (4-5 times) with lysis buffer

• Elute proteins by boiling in SDS sample buffer

• Analyze by Western blotting for potential interacting partners

Advantages of biotin-streptavidin approach:

• Higher binding affinity compared to antibody-protein A/G interactions

• Reduced background from heavy and light chains in western blot detection

• Possibility of gentle elution using biotin competition

For studying virus-induced interactions:

• Compare samples from uninfected and virus-infected cells

• Include time course analysis (0h, 6h, 12h, 24h post-infection)

• Focus on detecting MAVS and TRAF3 as known interaction partners

Research using similar approaches has demonstrated that TRAF3IP3 associates with MAVS upon viral infection and facilitates the recruitment of TRAF3 to this complex, which is critical for downstream signaling activation .

What methodological approaches can help resolve contradictory findings about TRAF3IP3 function?

To address contradictory findings regarding TRAF3IP3 function, researchers can employ systematic approaches using the Biotin-conjugated TRAF3IP3 antibody:

Cell type-specific analysis:

• Compare TRAF3IP3 expression and function across different immune cell subtypes

• Use the antibody for immunohistochemistry and flow cytometry to quantify expression levels

• Correlate expression with functional outcomes in each cell type

Context-dependent functional assessment:

• Design experiments testing TRAF3IP3 function under diverse stimulation conditions (viral infection, TLR ligands, cytokines)

• Track TRAF3IP3 localization and interaction partners under each condition

• Compare results between primary cells and cell lines to identify potential artifacts

Genetic validation:

• Use CRISPR/Cas9 to generate targeted TRAF3IP3 domain mutants

• Verify mutant expression using the antibody

• Assess which functions are affected by specific domain mutations

Research has shown that TRAF3IP3 has dual functions in antiviral immunity and B cell development through autophagy regulation . These seemingly distinct roles might reflect cell type-specific functions or context-dependent activation, which can be systematically investigated using the approaches outlined above.

What controls should I include when using TRAF3IP3 antibody in my experiments?

Including appropriate controls is essential for validating results obtained with the Biotin-conjugated TRAF3IP3 antibody:

Positive controls:

• Cell lines known to express high levels of TRAF3IP3 (e.g., immune cells)

• Recombinant TRAF3IP3 protein (particularly the region containing AA 74-185)

• TRAF3IP3 overexpression lysates

Negative controls:

• TRAF3IP3 knockout or knockdown samples

• Cell lines with naturally low TRAF3IP3 expression

• Primary antibody omission control (for immunostaining)

• Isotype control (rabbit IgG with biotin conjugation)

Specificity controls:

• Peptide competition/blocking experiments using the immunizing peptide (AA 74-185)

• Comparison with a different TRAF3IP3 antibody targeting another epitope

• Validation across multiple applications (WB, IF, ELISA) to confirm consistency

For viral infection studies, additional controls should include:

• Uninfected cells processed identically to infected samples

• Time course samples to capture dynamic changes

• UV-inactivated virus to distinguish between viral entry and replication effects

Research practices demonstrate that rigorous validation with these controls enhances data reliability and facilitates accurate interpretation of TRAF3IP3's functional roles in different experimental contexts .

What is the molecular mechanism of TRAF3IP3 in the MAVS-TRAF3 signaling axis?

The molecular mechanism by which TRAF3IP3 functions in the MAVS-TRAF3 signaling axis involves several key steps that can be studied using the Biotin-conjugated TRAF3IP3 antibody:

Sequential recruitment model:

• Upon RNA virus infection, RIG-I recognizes viral RNA and undergoes Riplet-mediated polyubiquitination

• Activated RIG-I promotes prion-like aggregation and activation of MAVS

• TRAF3IP3 accumulates on mitochondria and binds to multimerized MAVS-Region III

• TRAF3IP3 facilitates TRAF3 recruitment to MAVS

• TRAF3 recruitment leads to TBK1-IRF3 activation, resulting in type I interferon production

Key experimental evidence:

• TRAF3IP3 specifically affects TBK1-IRF3 activation but not IKK-NF-κB activation upon RNA virus infection

• TRAF3IP3 deficiency cripples RIG-I- and MAVS-triggered IFNB production but doesn't affect TBK1- or IRF3-triggered IFNB induction

• Full-length MAVS is required for TRAF3IP3-mediated enhancement of interferon production

Research has demonstrated that TRAF3IP3's role is specific to the MAVS-dependent pathway, as interferon induction via TBK1 or constitutively active IRF3 (S396D) remains unaffected in TRAF3IP3-deficient cells . This positioning in the signaling cascade makes TRAF3IP3 a critical regulatory node for antiviral immunity against RNA viruses.

How do I validate TRAF3IP3 antibody specificity in my experimental system?

Validating the specificity of the Biotin-conjugated TRAF3IP3 antibody in your experimental system requires a multi-faceted approach:

Genetic validation:

• Compare antibody reactivity in wild-type versus TRAF3IP3 knockout/knockdown samples

• Perform rescue experiments with TRAF3IP3 re-expression to restore antibody reactivity

• Test antibody against cells expressing truncated TRAF3IP3 variants to confirm epitope specificity

Biochemical validation:

• Perform peptide competition assays using the immunizing peptide (AA 74-185)

• Compare reactivity patterns with antibodies targeting different TRAF3IP3 epitopes

• Verify molecular weight of detected protein (64kDa) across different sample types

Application-specific validation:

• For Western blotting: Run gradient gels to confirm single band at expected molecular weight

• For immunofluorescence: Compare staining pattern with GFP-tagged TRAF3IP3 localization

• For immunoprecipitation: Confirm pulled-down protein by mass spectrometry

Experimental evidence indicates that properly validated antibodies show consistent detection of endogenous TRAF3IP3 with minimal background. Validation across multiple experimental systems (human vs. mouse) and applications enhances confidence in the specificity of antibody-based detection methods.

What phenotypes are observed in TRAF3IP3-deficient models and how does this inform antibody usage?

TRAF3IP3-deficient models exhibit several distinct phenotypes that can serve as validation benchmarks for antibody-based studies:

Immune cell development phenotypes:

• Significant reduction in common lymphoid progenitors (CLPs)

• Inhibition of B cell development in the bone marrow

• Complete absence of marginal zone B cells in the spleen

Functional immune deficiencies:

• Reduced serum natural antibodies

• Impaired T cell-independent type II (TI-II) responses to antigens like TNP-Ficoll

• Compromised antiviral immunity with increased susceptibility to RNA virus infection

Cellular mechanism defects:

• Diminished autophagy levels in B cells

• Increased apoptosis rates in marginal zone B cell populations

• Defective TRAF3 recruitment to MAVS during viral infection

These phenotypes provide valuable experimental contexts for antibody usage:

• Confirm antibody specificity by demonstrating absence of staining in knockout tissues

• Use antibody to track rescue of phenotypes in reconstitution experiments

• Apply antibody to detect compensatory changes in related proteins

Research has demonstrated that TRAF3IP3-deficient mice show attenuated interferon responses and severely crippled innate immunity to RNA virus infection , making these models valuable for validating antibody-based detection in antiviral immunity studies.

What are the key considerations when selecting between different TRAF3IP3 antibodies for specific applications?

When selecting between different TRAF3IP3 antibodies, including the Biotin-conjugated variant, researchers should consider several critical factors:

Epitope targeting:

• Different antibodies recognize distinct epitopes (e.g., AA 74-185, AA 231-330, or other internal regions)

• Epitope accessibility may vary across applications and experimental conditions

• Post-translational modifications might affect epitope recognition

Host species and clonality:

• Rabbit polyclonal antibodies (like the Biotin-conjugated TRAF3IP3 antibody) offer high sensitivity but batch-to-batch variation

• Monoclonal antibodies provide consistency but potentially lower sensitivity

• Host species affects compatibility with other antibodies in multi-color applications

Application-specific requirements:

• For Western blotting: Select antibodies validated for the 64kDa TRAF3IP3 protein

• For immunoprecipitation: Consider advantages of biotin conjugation for clean pull-downs

• For immunofluorescence: Choose antibodies proven to detect native protein conformation

Research precedent should guide selection, as antibodies with published track records in specific applications offer greater reliability. The biotin conjugation provides particular advantages for detection sensitivity and flexibility across multiple experimental platforms.