TRIM15 Antibody

What is TRIM15 and what are its key structural characteristics?

TRIM15 (Tripartite motif-containing protein 15) is a member of the TRIM/RBCC protein family that functions as an E3 ubiquitin-protein ligase. The protein consists of 465 amino acids with a calculated molecular weight of approximately 52 kDa. TRIM15 has also been identified under alternative names including RNF93, ZNF178, and ZNFB7. This protein is primarily localized in the nucleus and cytoplasm, and has significant roles in ubiquitination processes that regulate various cellular functions including immune responses and NF-kappaB signaling pathways .

What are the most effective applications for TRIM15 antibodies in research?

TRIM15 antibodies have demonstrated effectiveness across multiple experimental applications, with Western blot being the most widely used technique. Immunofluorescence (IF) and immunohistochemistry (IHC) represent other common applications that yield reliable results. When selecting an antibody for specific applications, researchers should consider validated antibodies with demonstrated reactivity in their experimental system. For instance, specific antibodies like 83582-5-RR have been validated for IF/ICC applications with optimal dilution ranges (1:125-1:500) . Co-immunoprecipitation (Co-IP) assays have also proven effective for studying TRIM15 protein interactions, particularly in investigating its binding partners such as Keap1 .

What are the most effective protocols for TRIM15 detection in cancer tissues?

For optimal TRIM15 detection in cancer tissues, immunohistochemistry represents a highly effective approach. Based on published methodologies, researchers should consider the following protocol elements: (1) Tissue fixation in 10% neutral-buffered formalin followed by paraffin embedding; (2) Antigen retrieval using citrate buffer (pH 6.0) with heat-induced epitope retrieval; (3) Blocking with 5% normal serum to reduce nonspecific binding; (4) Incubation with validated TRIM15 antibody at optimized dilution (typically 1:100-1:200) overnight at 4°C; (5) Signal development using appropriate detection systems; and (6) Counterstaining with hematoxylin. This approach has been successfully employed in studies examining TRIM15 expression in non-small cell lung cancer tissues, demonstrating significant correlations between expression levels and clinicopathological features .

How should researchers design Western blot experiments to accurately detect TRIM15?

For successful Western blot detection of TRIM15 (52 kDa), researchers should implement the following methodology: (1) Protein extraction using lysis buffers containing protease inhibitors to prevent degradation; (2) Protein separation on 8-12% SDS-PAGE gels with careful loading control selection (β-actin has proven effective); (3) Transfer to nitrocellulose membranes at optimal voltage/time settings; (4) Blocking with 5% non-fat dry milk or BSA in TBST; (5) Primary antibody incubation with validated TRIM15 antibodies overnight at 4°C; (6) HRP-conjugated secondary antibody incubation; and (7) Visualization using enhanced chemiluminescence reagents. This protocol has been successfully implemented in studies examining TRIM15 expression in cancer tissues, demonstrating clear band specificity at the expected molecular weight .

What are the best practices for co-immunoprecipitation studies involving TRIM15?

Co-immunoprecipitation (Co-IP) studies involving TRIM15 require careful optimization to maintain protein interactions while minimizing background. Based on published methodologies, researchers should: (1) Harvest cells in appropriate IP lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA with protease inhibitors); (2) Incubate lysates on ice for 40 minutes followed by centrifugation at 12,000×g at 4°C; (3) Pre-clear lysates with protein A/G agarose beads; (4) Incubate cleared lysates with anti-TRIM15 antibody (or interaction partner antibody) overnight with rotation; (5) Add protein A/G agarose beads and continue incubation; (6) Wash beads thoroughly with lysis buffer (at least 3 washes); and (7) Elute bound proteins for SDS-PAGE analysis. This approach has been successfully employed to identify interactions between TRIM15 and proteins such as Keap1 in cancer research .

What is the role of TRIM15 in tyrosine kinase inhibitor resistance in hepatocellular carcinoma?

TRIM15 plays a critical role in tyrosine kinase inhibitor (TKI) resistance in hepatocellular carcinoma (HCC) through a complex regulatory mechanism. Research has demonstrated that TRIM15 is abnormally upregulated in liver cancer cells following TKI treatment, and this upregulation contributes directly to resistance development. Mechanistically, this process involves the AKT/FOXO1 axis regulating TRIM15 expression. Once upregulated, TRIM15 acts as an E3 ligase to induce nuclear translocation of LASP1 by mediating its K63-linked polyubiquitination. This LASP1 translocation subsequently modulates TKI sensitivity through increased AKT phosphorylation and Snail expression. This forms a regulatory loop (AKT/FOXO1/TRIM15/LASP1) that perpetuates the resistance mechanism. Researchers investigating TKI resistance should consider TRIM15 as a potential therapeutic target, as disrupting this regulatory loop could potentially resensitize resistant cells to TKI treatment .

How does TRIM15 contribute to non-small cell lung cancer progression?

TRIM15 significantly contributes to non-small cell lung cancer (NSCLC) progression through multiple mechanisms centered on the Keap1-Nrf2 signaling pathway. Studies have demonstrated that TRIM15 is frequently upregulated in NSCLC samples compared to normal tissues, with expression levels correlating with poor prognosis. Functionally, TRIM15 promotes cancer cell proliferation and metastasis both in vitro and in vivo, dependent on its E3 ubiquitin ligase activity. The molecular mechanism involves TRIM15 directly targeting Keap1 (the principal regulator of Nrf2) for ubiquitination and degradation. This prevents Nrf2 degradation, leading to enhanced antioxidant response and tumor progression. Researchers studying NSCLC should consider TRIM15 expression analysis in their experimental designs, as it represents both a potential prognostic biomarker and therapeutic target. Methodologically, this pathway can be studied through combined approaches of TRIM15 knockdown/overexpression, ubiquitination assays, and downstream target analysis .

What methods are most effective for analyzing TRIM15 expression correlation with patient survival data?

For analyzing correlations between TRIM15 expression and patient survival outcomes, researchers should implement a multi-layered approach combining molecular and clinical data. The recommended methodology includes: (1) Initial analysis using bioinformatics tools like GEPIA (http://gepia.cancer-pku.cn/) to examine TRIM15 mRNA expression using TCGA and GTEx databases; (2) Validation through protein-level analysis using tissue microarray-based immunohistochemistry with standardized scoring systems; (3) Correlation of expression data with comprehensive clinicopathological parameters; and (4) Survival analysis using Kaplan-Meier methods with log-rank tests for statistical significance. This approach has successfully demonstrated significant associations between high TRIM15 expression and lower survival rates in NSCLC patients. Researchers should ensure adequate sample sizes and careful matching of clinical parameters to establish reliable prognostic correlations. When reporting results, hazard ratios with confidence intervals should be included alongside p-values to provide complete statistical context .

How can researchers effectively design ubiquitination assays to study TRIM15 E3 ligase activity?

To effectively study TRIM15 E3 ligase activity through ubiquitination assays, researchers should implement the following detailed methodology: (1) Establish experimental systems with tagged TRIM15 and potential substrate proteins (e.g., HA-tagged TRIM15 and His-tagged substrate); (2) Perform cell-based ubiquitination assays by co-transfecting TRIM15, substrate, and ubiquitin constructs; (3) Include appropriate controls including E3 ligase-dead TRIM15 mutants and empty vectors; (4) For increased specificity, use constructs expressing K48-specific or K63-specific ubiquitin to distinguish between degradative and non-degradative ubiquitination; (5) Following immunoprecipitation of the substrate, analyze ubiquitination patterns using specific antibodies against ubiquitin linkages (anti-K48-linkage or anti-K63-linkage specific polyubiquitin antibodies); and (6) Complement with in vitro ubiquitination assays using purified components to confirm direct E3 ligase activity. This comprehensive approach can effectively characterize the specific ubiquitination patterns mediated by TRIM15, as demonstrated in studies of TRIM15-mediated Keap1 ubiquitination .

What controls are essential when examining TRIM15 subcellular localization?

When examining TRIM15 subcellular localization using immunofluorescence techniques, researchers must implement several critical controls to ensure reliable interpretations: (1) Antibody specificity controls including TRIM15 knockout/knockdown cells to confirm signal specificity; (2) Peptide competition assays where available TRIM15 blocking peptides can verify antibody specificity; (3) Co-staining with established subcellular markers (e.g., DAPI for nucleus, phalloidin for actin cytoskeleton, paxillin for focal adhesions) to accurately determine localization patterns; (4) Inclusion of related TRIM family proteins as comparators to identify family-specific versus TRIM15-specific localization patterns; (5) Validation with multiple antibodies targeting different TRIM15 epitopes; and (6) Complementary approaches such as subcellular fractionation followed by Western blot to biochemically confirm localization patterns. These controls are particularly important given TRIM15's reported dual localization in both nuclear and cytoplasmic compartments, as well as its association with focal adhesion structures .

What techniques can researchers use to investigate the interaction between TRIM15 and focal adhesion components?

To comprehensively investigate interactions between TRIM15 and focal adhesion components, researchers should employ a multi-technique approach: (1) Co-immunoprecipitation assays using antibodies against TRIM15 and known focal adhesion proteins like paxillin, followed by mass spectrometry for unbiased protein complex identification; (2) Proximity ligation assays (PLA) to visualize and quantify protein-protein interactions in situ with spatial resolution; (3) Advanced microscopy techniques including FRET (Fluorescence Resonance Energy Transfer) or FLIM (Fluorescence-Lifetime Imaging Microscopy) to measure direct protein interactions in living cells; (4) Immunofluorescence co-localization with quantitative colocalization analysis using Pearson's or Mander's coefficients; (5) Live-cell imaging using fluorescently tagged proteins to monitor dynamic interactions during focal adhesion assembly/disassembly; and (6) Functional assays including TRIM15 depletion or overexpression followed by analysis of focal adhesion dynamics and cell migration parameters. This integrated approach has successfully identified TRIM15 as a component of focal adhesions that regulates their disassembly, providing important insights into its non-canonical functions beyond E3 ligase activity .

How can researchers address non-specific binding when using TRIM15 antibodies in immunohistochemistry?

Non-specific binding in TRIM15 immunohistochemistry can be systematically addressed through the following optimization strategies: (1) Implement extensive blocking protocols using a combination of 3-5% normal serum (matched to secondary antibody host species) with 0.1-0.3% Triton X-100 and 1% BSA; (2) Optimize primary antibody dilution through careful titration experiments (starting with manufacturer recommendations, typically 1:100-1:500); (3) Increase washing steps duration and frequency (at least 3×10 minutes with gentle agitation); (4) Include peptide competition controls where the antibody is pre-incubated with blocking peptide; (5) Utilize TRIM15 knockout/knockdown tissues as negative controls; (6) Consider alternative antigen retrieval methods if citrate buffer yields high background (try EDTA buffer at pH 9.0); and (7) If high background persists, implement biotin/avidin blocking steps when using biotin-based detection systems. These methodological refinements have proven effective in studies requiring specific TRIM15 detection in complex tissue environments such as hepatocellular carcinoma and lung cancer specimens .

What strategies can address contradictory results when studying TRIM15 function across different cancer types?

When facing contradictory results regarding TRIM15 function across different cancer types, researchers should implement a systematic approach to reconcile these differences: (1) Conduct comprehensive cell line validation studies to verify TRIM15 expression levels across the specific cancer models being used; (2) Perform rescue experiments with wild-type and mutant TRIM15 constructs to confirm phenotype specificity; (3) Carefully analyze tissue context differences that might explain differential functions (e.g., differential expression of TRIM15 substrates or cofactors); (4) Implement domain-specific mutants to identify which TRIM15 functional domains are relevant in each cancer context; (5) Consider temporal aspects of TRIM15 function by using inducible expression systems; (6) Examine potential post-translational modifications of TRIM15 that might differ between cancer types; and (7) Analyze potential alternative splicing of TRIM15 that might generate tissue-specific isoforms with altered functions. This comprehensive approach can help reconcile the seemingly contradictory roles reported for TRIM15 in hepatocellular carcinoma (where it contributes to tyrosine kinase inhibitor resistance) versus its role in non-small cell lung cancer (where it promotes tumor progression through Keap1-Nrf2 signaling) .

How should researchers quantify TRIM15 expression in tissue microarrays for accurate clinical correlations?

For accurate quantification of TRIM15 expression in tissue microarrays (TMAs) and subsequent clinical correlations, researchers should implement a rigorous, standardized methodology: (1) Use validated TRIM15 antibodies with demonstrated specificity in immunohistochemistry applications; (2) Implement standardized staining protocols with appropriate positive and negative controls on each TMA slide; (3) Establish a clear scoring system combining both intensity (0-3 scale: negative, weak, moderate, strong) and percentage of positive cells (resulting in H-scores or similar composite scores); (4) Ensure scoring is performed independently by at least two trained pathologists blinded to clinical outcomes; (5) Calculate inter-observer reliability using kappa statistics; (6) Establish clear, pre-defined cutoff values for categorizing expression levels (low vs. high) based on statistical methods such as X-tile or receiver operating characteristic curve analysis; and (7) Validate findings in independent patient cohorts whenever possible. This methodology has successfully identified significant correlations between high TRIM15 expression and aggressive tumor phenotypes in non-small cell lung cancer, demonstrating prognostic relevance .

What statistical approaches are most appropriate for analyzing TRIM15 expression correlation with clinicopathological features?

For robust statistical analysis of correlations between TRIM15 expression and clinicopathological features, researchers should implement a comprehensive approach: (1) Use chi-square or Fisher's exact tests for categorical variables (e.g., tumor grade, lymph node status); (2) Apply Student's t-test or Mann-Whitney U test for continuous variables depending on data distribution; (3) Implement multivariate analysis through Cox proportional hazards regression models to identify independent prognostic factors; (4) Calculate hazard ratios with 95% confidence intervals to quantify risk associations; (5) Conduct Kaplan-Meier survival analyses with log-rank tests to compare survival outcomes between TRIM15 expression groups; (6) Consider propensity score matching to minimize bias when comparing groups with imbalanced characteristics; and (7) Validate findings through bootstrapping or cross-validation approaches. When reporting results, researchers should clearly state both the statistical significance (p-value) and the effect size to provide a complete picture of the clinical relevance. This approach has demonstrated significant associations between high TRIM15 expression and lower survival rates in non-small cell lung cancer patients, establishing its potential as a prognostic biomarker .