TNFRSF1A Antibody

What is TNFRSF1A and why is it significant in research?

TNFRSF1A is a cell surface receptor for tumor necrosis factor-alpha (TNF-α) that plays critical roles in inflammatory signaling, cell survival, and apoptosis. Research significance stems from its involvement in various pathways including mTOR, JNK, IKK, caspase 3, MAPK, and NF-κB signaling . The receptor contains an extracellular domain that can be proteolytically cleaved, creating soluble forms that modulate TNF signaling. TNFRSF1A has been identified as a potential biomarker in liver injury and sepsis , and it represents a therapeutic target for osteoporosis, chronic inflammatory conditions, and autoimmune diseases . Recent research has also identified TNFRSF1A as a cancer marker in renal cell carcinoma .

What are the validated applications for TNFRSF1A antibodies?

TNFRSF1A antibodies have been validated for multiple research applications:

• Validated applications: Western Blot, Flow Cytometry, Immunocytochemistry, Immunohistochemistry, CyTOF, Agonist Activity assays

• Additional cited applications: ELISA Development, Functional Assays, Immunohistochemistry (both frozen and paraffin-embedded samples), Immunoprecipitation, and Neutralization assays

When selecting an antibody, researchers should confirm validation for their specific application, as performance can vary significantly between applications even with the same antibody.

How do polyclonal and monoclonal TNFRSF1A antibodies differ in research applications?

Polyclonal TNFRSF1A antibodies (like goat polyclonal IgG) recognize multiple epitopes on the target protein, potentially offering higher sensitivity but lower specificity . They are particularly useful for detecting proteins expressed at low levels or when protein conformation may be altered.

Monoclonal antibodies (such as clone #16805) recognize a single epitope, providing higher specificity but potentially lower sensitivity . They are preferable for applications requiring consistent lot-to-lot reproducibility and when distinguishing between closely related proteins.

For functional studies examining TNFRSF1A signaling, monoclonal antibodies with known epitope binding regions may be advantageous, especially when targeting specific functional domains or when agonistic/antagonistic activities are required .

How should TNFRSF1A antibodies be validated for Western blot applications?

Validation for Western blot applications should follow this methodological approach:

• Positive controls: Include recombinant human TNFRSF1A protein (such as catalog #636-R1) as a positive control

• Reduction conditions: Test under both reducing and non-reducing conditions, noting that some antibodies (e.g., clone #16805) may only detect TNFRSF1A under non-reducing conditions

• Expected molecular weight: Look for a band at approximately 55 kDa, but be aware that multiple bands may appear due to:

  • Oligomerization

  • Self-aggregation

  • Cleavage of the TNF-R1 extracellular domain

• Loading control: Include appropriate loading controls (e.g., β-Actin) to normalize protein quantities

• Concentration optimization: For polyclonal antibodies, start with 0.1 μg/mL concentration and adjust as needed based on signal-to-noise ratio

A properly validated Western blot protocol should demonstrate specificity through absence of signal in negative controls and appropriate molecular weight detection in positive controls.

What are the optimal conditions for flow cytometric detection of TNFRSF1A?

For optimal flow cytometric detection of TNFRSF1A:

• Antibody concentration: Use approximately 2.5 μg per 10^6 cells as a starting point

• Cell preparation: For human samples, peripheral blood monocytes or lymphocytes are appropriate cell types for TNFRSF1A detection

• Fixation: Use mild fixation protocols; harsh fixation may destroy or mask the epitope

• Controls: Include:

  • Isotype controls to assess non-specific binding

  • Unstained controls for autofluorescence calibration

  • FMO (fluorescence minus one) controls for accurate gating

• Compensation: Proper compensation is essential when using multiple fluorophores

• Permeabilization: Generally unnecessary for TNFRSF1A detection as it's primarily a cell surface receptor, but may be required for certain experimental designs

Researchers should validate protocols using cells known to express TNFRSF1A (such as activated T cells or monocytes) before proceeding with experimental samples.

How can I optimize immunocytochemistry protocols for TNFRSF1A detection?

For optimal immunocytochemistry detection of TNFRSF1A:

• Antibody concentration: Start with 5-15 μg/mL for polyclonal antibodies

• Fixation method: Use immersion fixation for suspension cells like peripheral blood lymphocytes

• Incubation conditions: Typical protocol includes:

  • Primary antibody incubation for 3 hours at room temperature or overnight at 4°C

  • Secondary antibody incubation for 1-2 hours at room temperature

• Counterstaining: Consider nuclear counterstaining (e.g., DAPI) for better visualization of cellular localization

• Controls: Include:

  • Negative controls (isotype or secondary-only)

  • Positive controls (cells known to express TNFRSF1A)

• Signal enhancement: If needed, consider tyramide signal amplification or similar methods for weak signals

For non-adherent cells, follow specialized protocols such as the "Fluorescent ICC Staining of Non-adherent Cells" protocol referenced in the literature .

How do I interpret conflicting TNFRSF1A antibody results between different applications?

When facing conflicting results across different applications:

• Epitope accessibility: Different applications expose different epitopes. For example, denaturation during Western blotting may destroy conformational epitopes recognized in flow cytometry. Some antibodies (e.g., clone #16805) work under non-reducing conditions only .

• Expression levels: Flow cytometry may detect surface expression while Western blot measures total protein. Discrepancies may reflect differences in localization rather than total expression.

• Cross-reactivity: Some antibodies show cross-reactivity with related proteins or across species. For example, anti-human TNFRSF1A antibodies may show varying degrees of cross-reactivity with mouse TNFRSF1A .

• Methodological validation: Confirm that each technique has been properly validated:

  • For Western blots: confirm protein size and specificity

  • For flow cytometry: verify with appropriate controls

  • For immunohistochemistry: include relevant positive and negative controls

• Antibody quality: Consider antibody degradation or lot-to-lot variability

When possible, validate findings using multiple antibodies targeting different epitopes of TNFRSF1A to increase confidence in your results.

Why might TNFRSF1A antibodies show unexpected cross-reactivity with other species?

Cross-reactivity with other species occurs for several methodological reasons:

• Epitope conservation: The peptide sequence corresponding to AA 20-43 of mouse TNF-R1 is identical to rat and human sequences in those residues, explaining why some antibodies show broad cross-reactivity .

• Functional homology: Some antibodies recognize functional domains that are conserved across species due to evolutionary pressure to maintain receptor function.

• Variable cross-reactivity by application: An antibody may demonstrate cross-reactivity in one application but not another. For example, anti-human TNFRSF1A antibodies exhibit TNF agonist activities on mouse L-929 fibroblast cells despite limited cross-reactivity in direct ELISAs (less than 20%) .

• Testing methodology: Cross-reactivity should be systematically evaluated through:

  • Direct ELISAs with recombinant proteins from different species

  • Functional assays on cell lines from different species

  • Sequence alignment analysis of the immunogen across species

When cross-reactivity is observed, researchers should determine whether it results from specific binding to conserved domains or non-specific interactions before utilizing the antibody in cross-species experiments.

What factors affect the reproducibility of functional assays using TNFRSF1A antibodies?

Several methodological factors influence reproducibility in functional assays:

• Antibody concentration: The effective dose (ED₅₀) for agonist activity in cytotoxicity assays is typically 1-6 μg/mL, but this can vary between experiments and cell lines .

• Cell line sensitivity: Different cell lines show variable sensitivity to TNFRSF1A-mediated effects. L-929 mouse fibrosarcoma cells are commonly used but require the metabolic inhibitor actinomycin D (1 μg/mL) for optimal sensitivity .

• Recombinant protein quality: When using recombinant TNFRSF1A (0.3 μg/mL) and TNF-alpha (0.25 ng/mL) in neutralization assays, protein quality and activity can significantly impact results .

• Incubation conditions: Temperature, duration, and media composition affect receptor-ligand interactions and downstream signaling.

• Cell passage number: High passage cells may show altered receptor expression or signaling capabilities.

• Readout methodology: Different viability or apoptosis assays have varying sensitivities and can produce inconsistent results.

To maximize reproducibility, researchers should standardize all experimental parameters, use low-passage cells, include appropriate controls, and perform multiple biological replicates.

How can TNFRSF1A antibodies be used in receptor neutralization and signaling studies?

For receptor neutralization and signaling studies:

• Neutralization approach:

  • Pre-incubate cells with anti-TNFRSF1A antibody (1-10 μg/mL) before TNF-alpha stimulation

  • Confirm neutralization by measuring downstream signaling events such as NF-κB phosphorylation

  • A dose-dependent inhibition curve should demonstrate specificity (as shown in studies where increasing concentrations of TNFR1AB (4, 6, and 10 μg/mL) inhibited TNF-alpha-induced phosphorylation of p105 and p65 in a dose-dependent manner)

• Signaling pathway analysis:

  • Monitor pathway-specific readouts such as:

    • Phosphorylation of NF-κB (p65, p105) via Western blot at 5 minutes post-stimulation

    • Activation of mTOR, JNK, IKK, caspase 3, and MAPK pathways

  • Use pathway-specific inhibitors in parallel to confirm specificity

• Functional consequences:

  • Assess cell-specific outcomes such as:

    • Cytotoxicity in sensitive cell lines like L-929

    • Expression of stemness markers (SSEA-4, OCT-4, NANOG) in stem cells

    • Inflammatory cytokine production

This approach allows detailed mapping of TNFRSF1A-dependent signaling cascades and their functional outcomes in various cellular contexts.

What are the considerations when using TNFRSF1A antibodies in single-cell analysis techniques?

For single-cell analysis of TNFRSF1A:

• Mass cytometry (CyTOF) applications:

  • Use CyTOF-ready antibodies without BSA or carrier proteins that could interfere with metal conjugation

  • Validate panel design to avoid signal overlap between channels

  • Consider fixation-sensitive epitopes that may be affected by sample processing

• Flow cytometry considerations:

  • Maintain consistent compensation settings across experiments

  • Assess TNFRSF1A expression heterogeneity within cell populations

  • Consider index sorting for linking protein expression to single-cell transcriptomics

• Single-cell RNA-seq correlation:

  • Validate antibody specificity before correlating protein expression with transcriptomic data

  • Consider that mRNA and protein levels may not directly correlate due to post-transcriptional regulation

  • Recent research on renal cell carcinoma utilized single-cell approaches to examine TNFRSF1A expression heterogeneity

• Spatial analysis:

  • Use validated immunofluorescence protocols to maintain epitope integrity

  • Consider multiplexing with other markers to identify specific cell populations expressing TNFRSF1A

These advanced techniques allow researchers to understand the heterogeneity of TNFRSF1A expression and its functional consequences at the single-cell level.

How can TNFRSF1A antibodies be utilized in studies of receptor shedding and soluble receptor dynamics?

To investigate TNFRSF1A shedding and soluble receptor dynamics:

• Detection of membrane-bound versus soluble forms:

  • Select antibodies that recognize either the extracellular domain (for both forms) or intracellular domain (for membrane-bound only)

  • Use Western blot to distinguish between the full-length (~55 kDa) and cleaved soluble forms

  • Different antibodies may preferentially detect specific forms or oligomerization states

• Shedding pathway analysis:

  • Monitor the iNOS-cGMP-TACE pathway implicated in TNFR1 shedding in hepatocytes following bacterial lipopolysaccharide stimulation

  • Use pharmacological inhibitors of metalloproteinases (e.g., TAPI-1) to block shedding

  • Measure soluble TNFRSF1A in culture supernatants by ELISA

• Functional implications:

  • Investigate how soluble TNFRSF1A modulates TNF signaling through neutralization assays

  • Examine the balance between pro-inflammatory (membrane-bound) and potentially anti-inflammatory (soluble) forms

  • Consider the role of soluble TNFRSF1A as a biomarker in conditions like sepsis-related liver injury

This approach helps elucidate the physiological and pathological roles of TNFRSF1A shedding in inflammatory regulation and disease progression.

How should researchers design experiments to investigate TNFRSF1A as a biomarker in liver injury and sepsis?

Based on current research on TNFRSF1A as a biomarker in sepsis-related liver injury :

• Tissue-specific analysis:

  • Compare TNFRSF1A expression across multiple tissues (liver, lung, kidney) to establish specificity

  • Use Western blot with anti-TNFRSF1A antibody and β-Actin as loading control

  • Correlate protein expression with histopathological changes (vacuolar degeneration, nuclear rupture)

• Animal models:

  • Establish appropriate sepsis models (e.g., cecal ligation and puncture)

  • Include time-course analysis to determine dynamic changes in TNFRSF1A expression

  • Consider comparing wildtype and TNFRSF1A-/- mice to assess functional significance

• Pathway investigation:

  • Examine relationships between TNFRSF1A and other signaling cascades:

    • mTOR and MAPK pathways (inflammatory regulation)

    • Caspase 3 and NF-κB pathways (apoptosis control)

  • Use specific antibodies against phosphorylated pathway components

• Clinical correlation:

  • Translate findings to human samples when possible

  • Correlate TNFRSF1A levels with clinical parameters of liver dysfunction

  • Consider measuring soluble TNFRSF1A in patient serum/plasma

This comprehensive approach can validate TNFRSF1A as a specific biomarker for septic liver injury and elucidate underlying mechanisms.

What methodological approaches are recommended for studying TNFRSF1A in cancer research?

For cancer research focused on TNFRSF1A :

• Single-cell heterogeneity analysis:

  • Apply single-cell RNA-seq and proteomics to analyze TNFRSF1A expression variability within tumors

  • Use validated antibodies for flow cytometry and immunohistochemistry to map expression at the protein level

  • Correlate with clinical parameters and patient outcomes

• Functional validation studies:

  • Design in vitro experiments to investigate cancer-promoting characteristics:

    • Proliferation assays following TNFRSF1A modulation

    • Migration and invasion assays

    • Apoptosis resistance measurements

  • Consider both antibody-based neutralization and genetic approaches (siRNA, CRISPR)

• Signaling pathway investigation:

  • Examine TNFRSF1A-mediated activation of:

    • NF-κB pathway (survival signaling)

    • MAPK pathway (proliferation)

    • Caspase cascade (apoptosis resistance)

  • Use phospho-specific antibodies for key pathway components

• Tumor microenvironment interaction:

  • Study how TNFRSF1A expression affects tumor-immune cell interactions

  • Investigate paracrine signaling between different cell populations

  • Consider multiplexed immunofluorescence or mass cytometry approaches

Particularly for renal cell carcinoma, both single-cell heterogeneity analysis and functional validation experiments have been employed to establish TNFRSF1A as a cancer marker .

How can researchers investigate TNFRSF1A as a therapeutic target in inflammatory and autoimmune diseases?

To investigate TNFRSF1A as a therapeutic target :

• Inhibition strategy development:

  • Use neutralizing antibodies at various concentrations (4-10 μg/mL) to block receptor function

  • Compare antibody-based approaches with soluble receptor mimetics or small molecule inhibitors

  • Assess differential effects on various signaling pathways

• In vitro disease models:

  • Establish relevant cell culture systems (e.g., synoviocytes for arthritis, hepatocytes for liver inflammation)

  • Measure inflammatory mediators following TNFRSF1A modulation

  • Assess functional outcomes such as:

    • Cell viability and proliferation

    • Inflammatory cytokine production

    • Tissue-specific functional parameters

• Selectivity assessment:

  • Compare effects of TNFRSF1A targeting versus other TNF receptors

  • Investigate potential off-target effects on related signaling pathways

  • Consider the balance between therapeutic efficacy and adverse effects

• Translational potential evaluation:

  • Assess pharmacokinetics and tissue distribution of antibody-based therapeutics

  • Determine optimal dosing regimens for sustained pathway modulation

  • Consider combination approaches with existing therapies

This systematic approach can identify the most promising strategies for targeting TNFRSF1A in conditions like osteoporosis, chronic inflammatory disorders, and autoimmune diseases .