TNFSF10 Antibody

What is TNFSF10 and what is its biological significance?

TNFSF10, also known as TNF-related apoptosis-inducing ligand (TRAIL) or Apo2L, is a 33-35 kDa type II transmembrane glycoprotein belonging to the tumor necrosis factor superfamily. It plays a crucial role in inducing apoptosis in transformed cell lines while generally sparing normal cells, suggesting an important function in tumor surveillance. TNFSF10 is bioactive as a homotrimer, with a unique structural feature being a zinc ion complexed by cysteine residues (Cys240 in mouse, Cys230 in human) that is critical for structural stability . TNFSF10 has been implicated in thymocyte apoptosis, erythrocyte precursor regulation, and various immune functions. Its ability to selectively induce apoptosis in cancer cells has made it a significant target for cancer therapeutic research .

What are the structural characteristics of TNFSF10 that researchers should consider when selecting antibodies?

TNFSF10 contains distinct domains that researchers should consider when selecting antibodies for specific applications. Mouse TNFSF10 consists of a 17 amino acid N-terminal intracellular domain, a 20 amino acid transmembrane domain, and a 253 amino acid extracellular domain . The protein functions as a homotrimer, with the TNF homology domain (amino acids 118-291 in mouse) being highly conserved across species. This domain shares 85% amino acid identity with rat TRAIL and 70% identity with human, bovine, and porcine TRAIL . The presence of a zinc ion bound by cysteine residues in each monomer is essential for structural stability and biological activity. When designing experiments, researchers should consider whether their antibody recognizes the membrane-bound or soluble form of TNFSF10, as proteolytic cleavage can release the soluble form (sTRAIL) with distinct biological activities .

How do species-specific variations in TNFSF10 impact antibody selection?

Species-specific variations in TNFSF10 significantly impact antibody selection for cross-species studies. Mouse and human TNFSF10 share approximately 70% amino acid identity within the TNF homology domain, which affects antibody cross-reactivity . For instance, some antibodies like the human TRAIL/TNFSF10 antibody (AF375) show less than 5% cross-reactivity with recombinant mouse TRAIL in direct ELISAs . Additionally, the receptor systems differ between species - humans have two receptors that transduce apoptotic signals (TRAIL R1/DR4 and TRAIL R2/DR5), while mice only have one (TRAIL R2/DR5) . Mice also express unique decoy receptors (DcTRAIL R1/TNFRSF23 and DcTRAIL R2/TNFRSF22) that differ structurally from human regulatory receptors TRAIL R3 and TRAIL R4 . When designing cross-species experiments, researchers should verify antibody specificity through validation assays and consider using species-specific antibodies like AF375 for human samples and AF1121 for mouse samples to ensure accurate results .

What are the optimal conditions for using TNFSF10 antibodies in Western blot applications?

For optimal Western blot results with TNFSF10 antibodies, several methodological considerations are essential. Based on validated protocols, researchers should:

• Sample preparation: Use appropriate cell lysis buffers that preserve TNFSF10's structure. For example, when analyzing TRAIL in 786-O human renal cell adenocarcinoma cell lines, reducing conditions were employed .

• Antibody concentration: Use 1 μg/mL of anti-human TNFSF10 antibody (e.g., AF375) for optimal detection .

• Membrane type: PVDF membranes are recommended for better protein retention and reduced background .

• Secondary antibody selection: Use a species-appropriate HRP-conjugated secondary antibody, such as Anti-Goat IgG Secondary Antibody (HAF017) when working with goat primary antibodies .

• Detection considerations: Be aware that TNFSF10 may appear as multiple bands, typically at approximately 27 and 30 kDa, reflecting different glycosylation states or processing forms .

• Buffer systems: Employ Immunoblot Buffer Group 1 or similar optimized buffer systems to minimize background and enhance specific signal detection .

Following these guidelines will help ensure specific detection of TNFSF10 protein in Western blot applications while minimizing non-specific binding and background issues.

How can I optimize TNFSF10 antibody staining for immunohistochemistry?

Optimizing TNFSF10 antibody staining for immunohistochemistry requires careful attention to several methodological parameters:

• Tissue preparation: Both frozen and paraffin-embedded sections can be used, but preparation protocols differ. For paraffin sections of human brain (cortex) or prostate cancer tissue, heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013) is recommended before antibody incubation .

• Antibody concentration: For human tissues, use 5-15 μg/mL of anti-human TNFSF10 antibody (AF375). For prostate cancer tissue specifically, 3 μg/mL has been validated to provide optimal staining .

• Incubation conditions: For robust staining in human brain sections, incubate the primary antibody overnight at 4°C. For prostate cancer tissue, 1 hour at room temperature is sufficient when using 3 μg/mL concentration .

• Detection systems: The Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; CTS008) provides excellent results for colorimetric detection. For enhanced sensitivity, consider the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004) .

• Counterstaining: Hematoxylin provides good contrast as a counterstain (blue) against the DAB (brown) detection of TNFSF10 .

• Controls: Include both positive and negative controls to validate staining specificity. For mouse tissues, similar protocols can be applied using mouse-specific antibodies like AF1121 .

This optimized approach will help visualize the cellular and subcellular localization of TNFSF10 in various tissue samples with high specificity and low background.

What strategies can be employed to confirm the specificity of TNFSF10 antibody binding?

Confirming antibody specificity is crucial for generating reliable research data. For TNFSF10 antibodies, employ these validation strategies:

• Cross-reactivity testing: Assess specificity through direct ELISAs against related proteins. For example, the human TNFSF10 antibody (AF375) shows less than 5% cross-reactivity with recombinant mouse TRAIL .

• Western blot molecular weight verification: TNFSF10 should appear at approximately 27 and 30 kDa under reducing conditions. Verify that your antibody detects bands of the expected size .

• Functional neutralization assays: Assess whether the antibody can neutralize TNFSF10-induced biological activities. The neutralization dose (ND₅₀) for anti-human TNFSF10 antibody is typically <140 ng/mL in the presence of 12 ng/mL Recombinant Human TRAIL/TNFSF10 .

• Tissue expression pattern verification: Compare staining patterns with known TNFSF10 expression profiles in various tissues. TNFSF10 has been validated in human brain cortex, prostate cancer tissue, and mouse thymus .

• Genetic approaches: Use RNA interference (siRNA targeting TNFSF10) or TNFSF10-knockout models to confirm signal absence when the target is depleted .

• Multiple antibody validation: Employ antibodies from different hosts or targeting different epitopes to confirm consistent staining patterns.

These rigorous validation methods ensure that experimental observations are genuinely attributable to TNFSF10 rather than non-specific interactions.

How can TNFSF10 antibodies be used to investigate autophagy-apoptosis crosstalk?

TNFSF10 antibodies serve as critical tools for investigating the complex relationship between autophagy and apoptosis:

• Monitoring protein dynamics: Use TNFSF10 antibodies in Western blot analysis to track changes in TNFSF10 expression levels during autophagy induction. This helps establish temporal relationships between TNFSF10 signaling and autophagy activation .

• Dual detection strategies: Combine TNFSF10 antibodies with markers of autophagy (e.g., MAP1LC3B, SQSTM1/p62) to simultaneously monitor both pathways. Research has shown that TNFSF10 treatment gradually reduces SQSTM1/p62 levels while increasing MAP1LC3B-II, indicating autophagy induction .

• Autophagy flux assessment: Perform TNFSF10 antibody-based detection in the presence and absence of lysosomal inhibitors (e.g., chloroquine) to distinguish between increased autophagosome formation and reduced degradation. This approach revealed that TNFSF10 genuinely increases MAP1LC3B-II production rather than simply suppressing its clearance .

• Protein interaction studies: Use TNFSF10 antibodies in immunoprecipitation experiments to investigate interactions between TNFSF10 signaling components and autophagy regulators. For example, immunoprecipitation studies have shown that TNFSF10 treatment reduces the interaction between BCL2L1 and BECN1, triggering autophagy .

• Manipulating autophagy pathways: Combine TNFSF10 antibody-based detection with autophagy inhibitors (3-methyladenine, wortmannin) or genetic approaches (siRNA targeting ATG7 or BECN1) to establish causal relationships. These studies demonstrated that blocking autophagy enhances TNFSF10-induced cell death .

This multifaceted approach has revealed that TNFSF10-induced autophagy functions as a cytoprotective mechanism against TNFSF10-induced apoptosis, highlighting the complex interplay between these pathways.

What methods can be employed to study TNFSF10-mediated signaling pathways?

Investigating TNFSF10-mediated signaling pathways requires sophisticated methodological approaches:

• Signaling protein profiling: Use TNFSF10 antibodies alongside antibodies against key signaling mediators (MAPK8, NFκB) to monitor pathway activation. Western blot analysis has shown that MAPK8 mediates TNFSF10-induced BCL2L1 decrease, while NFκB has different effects .

• Inhibitor-based pathway dissection: Combine TNFSF10 treatment with specific inhibitors of signaling modules (MAPK8 inhibitors, NFκB inhibitors) to delineate pathway contributions. This approach revealed that suppressing MAPK8, but not NFκB, restored BCL2L1 levels after TNFSF10 treatment .

• Protein degradation analysis: Track the kinetics of anti-apoptotic protein degradation following TNFSF10 treatment. Studies have demonstrated that TNFSF10 causes a slight decrease of anti-apoptotic proteins (BIRC2, BIRC3, XIAP, CFLAR), which becomes dramatically enhanced when autophagy is inhibited .

• Multi-pathway interaction studies: Investigate crosstalk between proteasomal and lysosomal degradation pathways in TNFSF10 signaling. Research indicates that anti-apoptotic proteins are regulated through proteasome and lysosome crosstalk in the context of TNFSF10 signaling .

• Genetic manipulation approaches: Use siRNA-mediated knockdown of pathway components to confirm their roles. Knockdown of ATG7 or BECN1 effectively potentiated TNFSF10-induced degradation of anti-apoptotic proteins, confirming autophagy's protective role .

These approaches collectively provide a comprehensive picture of how TNFSF10 activates multiple signaling pathways that determine cell fate decisions between survival and apoptosis.

How are TNFSF10 antibodies utilized in cancer research applications?

TNFSF10 antibodies enable several sophisticated cancer research applications:

• Differential expression profiling: Use immunohistochemistry with TNFSF10 antibodies to compare expression levels between normal and cancerous tissues. Studies have successfully employed anti-TNFSF10 antibodies to detect expression in human prostate cancer tissues and renal cell adenocarcinoma cell lines .

• Therapeutic response prediction: Analyze TNFSF10 expression patterns before and after treatment to identify potential biomarkers of therapeutic response. Immunohistochemical staining of cancer tissues can reveal patterns of TNFSF10 localization that may correlate with treatment outcomes .

• Resistance mechanism investigation: Use TNFSF10 antibodies to study how cancer cells evade TNFSF10-induced apoptosis. Research has identified that autophagy induction serves as a protective mechanism against TNFSF10-induced apoptosis in cancer cells .

• Signaling pathway analysis: Combine TNFSF10 antibodies with antibodies against anti-apoptotic proteins (BIRC2, BIRC3, XIAP, CFLAR) to investigate resistance mechanisms. Studies have shown that inhibiting autophagy dramatically decreases these anti-apoptotic proteins in TNFSF10-treated cells, potentially overcoming resistance .

• Functional neutralization studies: Use neutralizing TNFSF10 antibodies to block endogenous TNFSF10 activity and assess its contribution to tumor progression or regression. The neutralization capacity of anti-human TNFSF10 antibodies has been characterized in cell cytotoxicity assays .

These approaches highlight TNFSF10's complex role in cancer biology, where it can induce tumor cell apoptosis but may be counteracted by protective mechanisms like autophagy, informing potential therapeutic strategies that combine TRAIL-based therapies with autophagy inhibitors.

How should researchers address inconsistent results when using TNFSF10 antibodies across different cell lines?

When encountering inconsistent results with TNFSF10 antibodies across cell lines, implement this systematic troubleshooting approach:

• Cell-specific expression levels: Different cell lines express varying levels of TNFSF10 and its receptors. Verify baseline expression through qRT-PCR before antibody-based detection . Studies examining TRAIL in cancer cells revealed significant variation in both baseline expression and induction patterns.

• Post-translational modifications: TNFSF10 undergoes various modifications affecting antibody recognition. Western blot analysis of TNFSF10 typically reveals multiple bands (27 and 30 kDa), reflecting different glycosylation states . Ensure your experimental conditions preserve these modifications.

• Sample preparation optimization: Different cell lines may require modified lysis protocols. For adherent cells like 786-O renal cell adenocarcinoma, established protocols using Immunoblot Buffer Group 1 under reducing conditions yield reliable results .

• Receptor expression profiling: Characterize TRAIL receptor expression in your cell lines. Mouse cells express different receptor patterns than human cells, with mice having only one death receptor (TRAIL R2/DR5) versus two in humans (TRAIL R1/DR4 and TRAIL R2/DR5) .

• Signaling pathway variations: Cell lines differ in their autophagy and apoptosis pathway components. When studying TNFSF10-induced effects, simultaneously monitor autophagy markers (MAP1LC3B, SQSTM1) and apoptotic markers to identify cell line-specific response patterns .

• Antibody validation in each cell line: Perform siRNA knockdown of TNFSF10 in each cell line to confirm signal specificity. This approach verifies that observed signals genuinely represent TNFSF10 rather than non-specific binding .

This comprehensive approach addresses the biological and technical variables underlying inconsistent results, leading to more reproducible findings across diverse cell models.

What factors contribute to variability in TNFSF10 antibody staining patterns in tissue samples?

Several factors contribute to variability in TNFSF10 immunohistochemical staining patterns:

• Tissue fixation and processing: Different fixation methods significantly impact epitope preservation. For TNFSF10 detection in paraffin-embedded human brain sections, heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013) is essential for optimal staining .

• Tissue-specific expression patterns: TNFSF10 shows distinct expression patterns across tissues. In human brain cortex, specific cellular staining patterns differ from those observed in prostate cancer tissue or mouse thymus . Compare your results with established expression patterns for proper interpretation.

• Pathological state influence: Disease states alter TNFSF10 expression. In osteoarthritis models, TNFSF10 expression increases in articular cartilage following destabilization of the medial meniscus (DMM) surgery compared to sham-operated controls .

• Antibody concentration optimization: Different tissues require different antibody concentrations. For human brain sections, 15 μg/mL of anti-human TNFSF10 antibody is recommended, while prostate cancer tissue requires only 3 μg/mL for optimal results .

• Detection system sensitivity: The choice of detection system affects staining intensity and pattern. For enhanced sensitivity in prostate cancer tissue, the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004) provides superior results compared to standard detection methods .

• Incubation conditions: Staining patterns vary with incubation protocols. Overnight incubation at 4°C yields different results compared to 1-hour incubation at room temperature .

Understanding these variables allows researchers to standardize protocols and correctly interpret variations in TNFSF10 staining patterns across different experimental contexts.

How should researchers interpret contradictory findings in studies of TNFSF10-mediated cell death mechanisms?

Interpreting contradictory findings in TNFSF10-mediated cell death research requires a nuanced analytical approach:

This comprehensive analytical framework enables researchers to reconcile apparently contradictory findings into a coherent understanding of TNFSF10's complex biological roles.

What are the emerging applications of TNFSF10 antibodies in studying inflammatory diseases?

TNFSF10 antibodies are increasingly utilized in inflammatory disease research, revealing complex roles beyond cancer biology:

• Osteoarthritis pathogenesis: Studies using TNFSF10 antibodies have identified TNFSF10 as a critical mediator in osteoarthritis progression. Immunohistochemical analysis demonstrated increased TNFSF10 expression in articular cartilage following destabilization of the medial meniscus (DMM) surgery compared to sham-operated controls, correlating with disease severity .

• Chondrocyte apoptosis regulation: TNFSF10 antibody-based detection revealed that IL-1β-induced collagenase activity increases TNFSF10 expression in articular chondrocytes, promoting apoptosis. This pathway involves OSCAR (osteoclast-associated receptor) co-stimulation of IL-1β signaling, representing a novel therapeutic target .

• Inflammatory signaling network mapping: Combining TNFSF10 antibodies with pathway-specific inhibitors has helped map inflammatory signaling networks. Research showed that OSCAR deficiency attenuates TRAIL-induced articular chondrocyte apoptosis, establishing a mechanistic link between inflammatory signaling and cell death in osteoarthritis .

• Homeostatic regulation in immune tissues: Immunohistochemical detection of TNFSF10 in mouse thymus has revealed its role in immune homeostasis. TNFSF10 antibody staining patterns in thymic tissue sections suggest involvement in thymocyte selection and development .

• Osteoprotegerin (OPG) interaction studies: The relationship between TNFSF10 and OPG represents an important inflammatory regulatory mechanism. Immunohistochemical analyses showed inverse correlation between TNFSF10 and OPG expression in articular cartilage during osteoarthritis progression .

These emerging applications highlight TNFSF10's multifaceted roles in inflammatory regulation, extending well beyond its traditional characterization as an apoptosis-inducing ligand in cancer research.

How do researchers integrate TNFSF10 antibody-based findings with genetic and genomic data?

Integrating TNFSF10 antibody-based findings with genetic and genomic approaches creates a comprehensive research framework:

• Gene expression correlation analysis: Researchers combine TNFSF10 protein detection (using antibodies) with transcriptomic profiling to identify correlation patterns. Studies have employed Venn diagrams to identify genes attenuated in articular cartilage of Oscar-deficient mice after DMM surgery, revealing 1,270 common genes potentially involved in TNFSF10-mediated osteoarthritis pathogenesis .

• Pathway enrichment integration: Antibody-based findings can be contextualized through pathway enrichment analysis of genomic data. Hypergeometric tests using hallmark gene annotations in MsigDB yielded enrichment scores for functional annotations associated with TNFSF10-regulated genes, providing mechanistic insights .

• Network visualization approaches: Researchers visualize complex relationships between TNFSF10 signaling components using tools like Cytoscape. This approach revealed simplified apoptotic signaling pathways altered by Oscar deficiency, with each gene colored according to its differential expression score (DIF) .

• Validation of genomic findings with protein-level data: TNFSF10 antibodies confirm protein-level changes predicted by genomic analyses. qRT-PCR analyses of TRAIL in osteoarthritic cartilage were confirmed at the protein level through immunohistochemistry, validating genomic findings .

• Multi-omics data integration: Advanced research integrates antibody-based protein detection with genomic, transcriptomic, and functional data. This approach has established that OSCAR regulates osteoarthritis pathogenesis via TRAIL-induced articular chondrocyte apoptosis, a finding supported by multiple data types .

This integrated approach provides robust, multi-level evidence for TNFSF10's biological roles, leading to more comprehensive understanding than possible with any single methodology.