TRAF7 Antibody

What is TRAF7 and why is it significant in research?

TRAF7 (TNF receptor-associated factor 7) is a 74.6 kDa protein that may also be known by several alternative names including CAFDADD, RFWD1, RNF119, E3 ubiquitin-protein ligase TRAF7, and RING finger and WD repeat-containing protein 1 . Unlike other TRAF family members, TRAF7 contains seven WD40 repeats at its C-terminal end, while still maintaining the characteristic RING finger domain (aa 125-160) and adjacent zinc finger domain (aa 221-287) at its N-terminal end . TRAF7 plays significant roles in multiple signaling pathways, particularly in regulating innate immune responses to viral infections by promoting K48-linked polyubiquitination of TBK1, thereby inhibiting cellular antiviral responses . The protein's involvement in these critical signaling cascades makes it an important target for research into immune function, cancer, and infectious disease mechanisms.

What types of TRAF7 antibodies are currently available for research applications?

Current research indicates multiple TRAF7 antibody products are available across at least 22 suppliers, offering both polyclonal and monoclonal options . Available antibodies include unconjugated formats primarily used in applications such as Western blotting, immunohistochemistry, ELISA, immunoprecipitation, and immunofluorescence . For example, Proteintech offers a polyclonal rabbit antibody (11780-1-AP) that has been validated for multiple applications including WB, IHC, IF, CoIP, and ELISA with demonstrated reactivity against human samples and predicted reactivity with other species . When selecting an antibody, researchers should consider the specific epitope targeted, whether it recognizes the N-terminal or other regions of TRAF7, as this may affect detection based on protein domains involved in specific protein-protein interactions.

How does antibody selection impact experimental outcomes when studying TRAF7?

Antibody selection fundamentally impacts experimental outcomes through specificity, sensitivity, and application compatibility. When studying TRAF7, researchers should first validate antibodies for their specific application through appropriate controls, as demonstrated in studies where researchers verified TRAF7 antibodies by both immunofluorescence and immunoblot analysis prior to investigating TRAF7 recruitment during bacterial infection . When selecting antibodies, researchers should consider: (1) the specific epitope recognized, as different domains of TRAF7 (RING finger, zinc finger, or WD40 repeats) may be differentially accessible depending on protein conformation or interaction partners; (2) species reactivity, as studies with orthologs in canine, porcine, monkey, mouse, and rat models require cross-reactive antibodies ; and (3) experimental application requirements, as some antibodies perform better in certain applications (fixed samples vs. native proteins). Comprehensive validation is particularly important when TRAF7 undergoes post-translational modifications or forms protein complexes that might mask epitopes.

What are the optimal protocols for detecting TRAF7 using Western blotting?

For optimal Western blot detection of TRAF7, researchers should consider the following methodological approach based on published protocols: First, prepare protein samples by fractionating via SDS-PAGE, considering that TRAF7 has an expected molecular weight of 67-75 kDa . For antibody selection, successful detection has been reported using rabbit anti-TRAF7 antibodies at 1:500 dilution (Proteintech) or equivalent alternatives . For protein loading controls, studies have employed antibodies against housekeeping proteins such as β-actin or β-tubulin at 1:1000 dilution . When analyzing complex formation or activation states, native PAGE can be performed using 7.5% acrylamide gels without SDS, as demonstrated in studies examining TRAF7's role in immunological signaling . To validate specificity, knockout/knockdown controls are highly recommended, as multiple studies have employed CRISPR-Cas9-generated TRAF7-deficient cell lines for antibody validation and functional studies . The observed molecular weight of TRAF7 typically ranges from 67-75 kDa, which aligns with the calculated molecular weight of 75 kDa .

How can TRAF7 be effectively visualized in cells using immunofluorescence microscopy?

For effective immunofluorescence visualization of TRAF7, researchers should follow this optimized protocol based on published methodologies: Begin by growing cells (e.g., MCF7 or HeLa) on appropriate coverslips or chamber slides . After experimental treatments, fix cells with 4% paraformaldehyde for 10 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 for 10 minutes . Block non-specific binding with 1% BSA for 60 minutes before proceeding to primary antibody incubation . For endogenous TRAF7 detection, validated antibodies such as those from Proteintech have shown successful visualization at recommended dilutions (typically 1:20-1:200 for IHC applications, with similar ranges for IF) . When studying co-localization with binding partners, dual labeling can be performed using appropriate secondary antibodies with distinct fluorophores (e.g., Alexa Fluor 488 and 647) . After counterstaining nuclei with DAPI (10 μg/mL for 10 minutes), images can be acquired using confocal microscopy . Research has shown that endogenous TRAF7 localizes to the nucleus, cytosol, and cytoplasmic puncta, with puncta formation becoming particularly prominent upon overexpression .

What are the recommended approaches for immunoprecipitation of TRAF7 and its interacting partners?

For successful immunoprecipitation of TRAF7 and its binding partners, researchers should implement the following methodological approach: Begin with efficient cell lysis in a buffer that preserves protein-protein interactions while effectively extracting TRAF7 from its various cellular compartments (nuclear, cytosolic, and membrane-associated pools) . For co-immunoprecipitation of transfected constructs, expression vectors encoding tagged versions of TRAF7 (HA-TRAF7 or FLAG-TRAF7) have been successfully employed to facilitate pull-down with anti-tag antibodies . When investigating native interactions, verified TRAF7 antibodies that have been validated for immunoprecipitation applications should be used, such as those referenced in published studies . To identify novel interaction partners, affinity purification coupled with mass spectrometry (AP-MS) has proven effective, as demonstrated in studies that identified MEKK2 as a TRAF7 interacting protein . For validation of specific interactions, yeast two-hybrid systems have also been successfully employed, as seen in studies investigating TBK1-TRAF7 interactions . When studying domain-specific interactions, researchers have successfully employed truncated constructs containing specific domains, such as the WD40 domain of TRAF7, to map interaction interfaces with binding partners like Tri1 .

How can CRISPR-Cas9 gene editing be optimized for TRAF7 knockout studies?

For optimized CRISPR-Cas9 gene editing of TRAF7, researchers should implement the following methodological approach based on published protocols: Begin with careful gRNA design targeting functionally critical regions of TRAF7, such as the conserved RING domain which is essential for its ubiquitin ligase activity . A validated gRNA sequence (5'-GCTACAACCGCTTCTCCGGG-3') has been successfully employed for TRAF7 targeting in published studies . For delivery, lentiviral vector systems such as lenti-CRISPR-V2 have demonstrated efficacy, allowing for stable integration and expression of Cas9 and gRNA . After transfection with appropriate packaging vectors (psPAX2 and pMD2G), collect viral supernatants at 36 hours post-transfection for subsequent infection of target cells . Following infection, implement a two-stage selection process: first, apply puromycin selection (1 μg/mL) for approximately seven days to eliminate non-transduced cells, then perform single-cell sorting into 96-well plates to establish monoclonal TRAF7-deficient cell lines . Validate knockout efficiency through multiple approaches including Western blot analysis using validated TRAF7 antibodies, genomic sequencing of the target region, and functional assays measuring altered cellular responses to stimuli such as viral infection .

What functional assays are most informative for studying TRAF7's role in cellular signaling pathways?

The most informative functional assays for studying TRAF7's signaling roles focus on its documented functions in immune regulation and protein-protein interactions. For investigating TRAF7's role in antiviral responses, researchers should employ viral infection models using VSV (Vesicular Stomatitis Virus) at appropriate MOIs (e.g., 0.2), followed by plaque assays to quantify viral replication in TRAF7-deficient versus control cells . To assess TRAF7's impact on the RLR signaling pathway, measure type I interferon production through ELISA of culture supernatants and qPCR analysis of IFN-β and other antiviral genes following viral stimulation . For analyzing TRAF7's E3 ubiquitin ligase activity, ubiquitination assays using co-expression of HA-tagged ubiquitin with TRAF7 and potential substrates like TBK1 can reveal the type of ubiquitin linkages (K48 versus K63) that TRAF7 catalyzes, which determines whether tagged proteins are degraded or activated . To investigate TRAF7's role in protein complex formation, co-immunoprecipitation followed by Western blotting for known or suspected interaction partners provides valuable insights into how TRAF7 scaffolds signaling complexes . Finally, for studying TRAF7's impact on transcription factor activation, native PAGE analysis of IRF3 dimerization and nuclear translocation of NF-κB components following appropriate stimuli provides a direct measure of downstream signaling events regulated by TRAF7 .

How does TRAF7 influence the recruitment and ubiquitination of TBK1 in innate immune signaling?

TRAF7 acts as a critical negative regulator of innate immune signaling by directly targeting TANK-binding kinase 1 (TBK1), a nodal protein in multiple signal transduction pathways . Mechanistically, TRAF7 binds directly to TBK1 through specific protein-protein interactions that have been validated through co-immunoprecipitation and yeast two-hybrid screening . Following binding, TRAF7 utilizes its RING domain to catalyze K48-linked polyubiquitination of TBK1, which marks TBK1 for proteasomal degradation . This ubiquitination is dependent on the conserved cysteine residue at position 131 within TRAF7's RING domain, as demonstrated through mutational analysis . The functional consequence of this interaction is reduced TBK1 protein levels, which impairs the activation of downstream transcription factors IRF3 and NF-κB, ultimately decreasing type I interferon production in response to viral infection . Experimentally, TRAF7 knockout enhances IRF3 activation and increases transcript levels of antiviral genes following stimulation, confirming TRAF7's inhibitory role in the pathway . This regulatory mechanism represents a potential homeostatic control point that prevents excessive inflammatory responses during viral infection. The detailed molecular mechanism can be studied through a combination of co-immunoprecipitation, ubiquitination assays, confocal microscopy for protein co-localization, and functional readouts of IRF3 activation and interferon production .

How does Chlamydia trachomatis exploit TRAF7 during infection?

Chlamydia trachomatis has evolved a sophisticated mechanism to subvert host immunity by targeting TRAF7 through the bacterial inclusion membrane protein Tri1 . During infection, Tri1 specifically interacts with host TRAF7 through its predicted coiled-coil domain, which binds to the WD40 domain of TRAF7 . This interaction results in the recruitment of TRAF7 to the chlamydial inclusion, as visualized by immunofluorescence microscopy using validated TRAF7 antibodies . Functionally, Tri1 competes with and displaces native TRAF7 binding partners, specifically mitogen-activated protein kinase kinase kinase 2 (MEKK2) and MEKK3, which are important signaling molecules in innate immune pathways . Through structure-function analysis using truncated variants of Tri1, researchers demonstrated that the coiled-coil domain (approximately amino acids 84-147) is both necessary and likely sufficient for TRAF7 binding . This molecular mimicry allows the bacteria to alter TRAF7-dependent signaling during infection, potentially modulating host immune responses to create a favorable environment for bacterial survival and replication . This mechanism highlights the evolutionary arms race between pathogens and host immunity and demonstrates how studying pathogen-host protein interactions can reveal novel aspects of cellular protein function.

What is the role of TRAF7 in cancer progression and tumor biology?

While the search results do not provide comprehensive information on TRAF7's role in cancer, there are important indications of its relevance in tumor biology. The search results mention that TRAF7 "is mutated in a subset of tumors" , suggesting potential tumor suppressor or oncogenic functions. TRAF7 antibodies have been successfully used in immunohistochemistry applications with human colon cancer tissue, indicating expression or altered localization in this cancer type . The protein's established roles in regulating key signaling pathways including NF-κB, JNK-AP-1, and interferon responses suggest potential mechanisms by which TRAF7 alterations could contribute to oncogenesis . As an E3 ubiquitin ligase that targets signaling molecules like TBK1 for degradation, mutations affecting TRAF7's enzymatic activity could potentially lead to dysregulated signaling cascades and altered cellular responses to environmental stimuli . For researchers investigating TRAF7 in cancer contexts, methodological approaches should include: (1) immunohistochemical analysis of tumor tissues using validated antibodies with appropriate antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0); (2) correlation of expression patterns with clinical parameters; (3) functional studies in cancer cell lines with TRAF7 knockout or overexpression; and (4) analysis of TRAF7 mutation patterns across cancer types using publicly available genomic databases .

How can background and non-specific binding be minimized when using TRAF7 antibodies in immunostaining applications?

To minimize background and non-specific binding when using TRAF7 antibodies in immunostaining, researchers should implement several optimization strategies based on published protocols. First, antibody validation is crucial—researchers should verify specificity through proper controls including TRAF7 knockout cells, as demonstrated in published studies . For immunohistochemistry applications, antigen retrieval methods significantly impact results, with recommendations to use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 as specified for validated TRAF7 antibodies . Optimization of antibody dilution is essential, with successful IHC applications reported at dilutions ranging from 1:20 to 1:200, requiring titration in each specific testing system . For difficult samples, extended blocking periods (60+ minutes) with 1-5% BSA have proven effective in reducing non-specific binding . When performing co-staining experiments, careful selection of compatible secondary antibodies is necessary to avoid cross-reactivity . For tissues with high endogenous peroxidase activity, additional quenching steps may be necessary before primary antibody application. Finally, sample-dependent optimization is often required, as noted in the antibody product literature . When troubleshooting, researchers should systematically adjust one parameter at a time while maintaining appropriate positive and negative controls to identify the specific source of background issues.

What are the critical considerations for validating a new lot of TRAF7 antibody?

Validating a new lot of TRAF7 antibody requires a systematic approach addressing specificity, sensitivity, and application compatibility. Begin with basic Western blot validation against positive control lysates containing endogenous TRAF7, comparing signal intensity, background, and molecular weight recognition (67-75 kDa) against the previous lot . Include negative controls such as TRAF7 knockout cell lysates generated through CRISPR-Cas9, as described in published methods (using validated gRNA sequence: 5′-GCTACAACCGCTTCTCCGGG-3′) . For application-specific validation, test the new lot in all intended applications following established protocols; for instance, if using for immunofluorescence, verify the expected localization patterns of TRAF7 to nucleus, cytosol, and cytoplasmic puncta as previously documented . Peptide competition assays can provide additional specificity confirmation by demonstrating signal reduction when the antibody is pre-incubated with immunizing peptide. For quantitative applications, construct a standard curve to assess linearity of detection across a concentration range. When validating for co-immunoprecipitation applications, verify the ability to pull down known interaction partners such as TBK1 or MEKK2/3 . Finally, cross-reference validation results against published literature and manufacturer data to ensure consistency with established reagent performance characteristics before implementing the new lot in critical experiments.

What emerging technologies could enhance our understanding of TRAF7's functional roles?

Emerging technologies offer promising approaches to deepen our understanding of TRAF7's multifaceted roles in cellular signaling. Proximity labeling techniques such as BioID or APEX2 fusion proteins could map the complete TRAF7 interactome in various cellular compartments, potentially revealing novel binding partners beyond the currently identified TBK1, MEKK2, and MEKK3 . CRISPR-based screens targeting TRAF7 interactors could systematically identify synthetic lethal interactions and functional redundancies in TRAF7 signaling networks. Advanced live-cell imaging using split fluorescent protein complementation could visualize TRAF7 interactions in real-time during cellular responses to stimuli such as viral infection or cytokine treatment . Single-cell transcriptomics and proteomics comparing wild-type and TRAF7-deficient cells could reveal cell-type specific roles and heterogeneity in TRAF7-dependent responses. Structural studies using cryo-electron microscopy could elucidate the molecular mechanisms of TRAF7's interactions with binding partners and substrates, particularly focusing on how the unique WD40 repeats of TRAF7 contribute to specificity . Finally, development of small molecule inhibitors or activators specifically targeting TRAF7's E3 ligase activity could provide valuable chemical biology tools to modulate TRAF7 function in experimental and potentially therapeutic contexts.

How might TRAF7-targeted therapies be developed for infectious or inflammatory diseases?

Development of TRAF7-targeted therapies represents a promising frontier given its established roles in regulating innate immunity and inflammatory signaling. Since TRAF7 negatively regulates the RLR signaling pathway by promoting K48-linked polyubiquitination of TBK1, inhibiting TRAF7 could potentially enhance antiviral responses in certain contexts . Conversely, in conditions characterized by excessive inflammation, augmenting TRAF7 activity might dampen harmful immune hyperactivation. Therapeutic strategies could include: (1) Small molecule inhibitors targeting the RING domain's E3 ligase activity, focusing specifically on the functionally critical cysteine residue at position 131 ; (2) Peptide-based disruptors of protein-protein interactions, particularly those targeting the interface between TRAF7's WD40 domain and specific binding partners ; (3) Gene therapy approaches to modulate TRAF7 expression in specific tissues; or (4) Pathogen-inspired strategies leveraging the mechanism by which bacterial Tri1 protein modulates TRAF7 function through displacement of endogenous binding partners . Development of such therapeutics would require comprehensive understanding of TRAF7's tissue-specific functions and careful evaluation of potential off-target effects, given its involvement in multiple signaling pathways. Methodologically, high-throughput screening of compound libraries against recombinant TRAF7 E3 ligase activity, followed by cellular validation in TRAF7-dependent functional assays, represents a practical approach for identifying candidate molecules for further development.