TNFRSF1A Antibody, Biotin conjugated

What is TNFRSF1A and what biological systems express this receptor?

TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A), also known as TNF-RI, CD120a, or p55, functions as a receptor for both TNF-alpha and TNF-beta cytokines. It occurs in both membrane-bound and soluble forms and plays critical roles in inflammation, apoptosis, and immune regulation pathways . TNFRSF1A is widely expressed across multiple human and murine tissue types, with particularly notable expression in certain cancer cell lines like the HL-60 human promyelocytic leukemia cells, which are often used as a source for TNF binding protein purification . The receptor contains extracellular domains that interact with ligands and intracellular domains that initiate downstream signaling cascades through NF-κB activation, as demonstrated in studies with dental pulp stem cells .

What experimental applications are suitable for biotin-conjugated TNFRSF1A antibodies?

Biotin-conjugated TNFRSF1A antibodies are versatile reagents applicable to multiple experimental techniques. They can be effectively utilized in Western Blotting (WB) for protein detection, Flow Cytometry (FACS) for cell surface receptor quantification, and various immunohistochemistry approaches including frozen tissue sections (IHC) . The biotin conjugation provides signal amplification advantages through subsequent streptavidin-based detection systems. For immunoprecipitation (IP) experiments, these antibodies can efficiently isolate TNFRSF1A protein complexes from cell lysates . Additionally, they perform well in immunofluorescence (IF) and immunocytochemistry (ICC) applications, allowing researchers to visualize receptor localization in fixed cells or tissues .

How should I validate the specificity of TNFRSF1A antibody in my experimental system?

Validating antibody specificity requires multiple complementary approaches:

• siRNA knockdown verification: Transfect cells with siRNAs targeting TNFRSF1A (as demonstrated with siRNA1, siRNA2, siRNA3 in U87 and U251 glioma cell lines) and verify reduced antibody signal via Western blot and qRT-PCR .

• Recombinant protein controls: Use purified recombinant TNFRSF1A protein as a positive control in Western blots, comparing migration patterns with your experimental samples .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with related proteins (particularly TNFRSF1B/TNF-RII) using sandwich immunoassays that can distinguish between specific binding and non-specific interactions .

• Molecular weight verification: TNFRSF1A typically appears at approximately 55 kDa on Western blots, though oligomerization, self-aggregation, or cleavage of the extracellular domain may produce additional bands that should be characterized .

• Functional validation: Confirming that the antibody blocks TNF-α-induced cellular responses (like NF-κB phosphorylation) provides functional validation of specificity .

What are the optimal sample preparation techniques for TNFRSF1A detection?

Sample preparation significantly impacts TNFRSF1A detection quality. For cell culture samples, harvest cells at 70-80% confluence to ensure optimal receptor expression levels. Lysis should be performed using buffers containing appropriate protease inhibitors to prevent receptor degradation. When preparing tissue samples, rapid freezing in liquid nitrogen followed by mechanical homogenization yields superior results compared to chemical extraction methods. For membrane proteins like TNFRSF1A, detergent selection is critical—RIPA buffer containing 0.1-0.5% NP-40 or Triton X-100 effectively solubilizes membrane-bound receptors while preserving antibody epitopes . For flow cytometry applications, gentle enzymatic dissociation methods (using collagenase rather than trypsin) better preserve cell surface TNFRSF1A epitopes. Importantly, avoid repeated freeze-thaw cycles of prepared samples as this can lead to protein degradation and epitope masking.

How can I optimize Western blot protocols specifically for TNFRSF1A detection?

Optimizing Western blot protocols for TNFRSF1A requires attention to several critical parameters:

• Sample preparation: Use RIPA buffer supplemented with phosphatase inhibitors and protease inhibitors to effectively extract TNFRSF1A while preserving its phosphorylation state. Avoid boiling samples for more than 5 minutes as this can cause receptor aggregation.

• Gel selection: 10-12% polyacrylamide gels provide optimal resolution for the ~55 kDa TNFRSF1A protein. Use gradient gels (4-20%) when analyzing both monomeric and oligomeric forms simultaneously .

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C yields optimal results for this membrane protein.

• Blocking conditions: 5% non-fat dry milk in TBST is generally effective, though 3% BSA may provide lower background for biotin-conjugated antibodies.

• Antibody dilution: The optimal working dilution for biotin-conjugated TNFRSF1A antibody typically ranges from 1:500 to 1:2000, but should be empirically determined for each lot .

• Detection system: Streptavidin-HRP conjugates (1:5000-1:10000) followed by enhanced chemiluminescence provide sensitive detection of biotinylated antibodies. Allow 45-60 minutes for streptavidin binding at room temperature.

• Stripping and reprobing: If necessary, use mild stripping buffers (50mM glycine, pH 2.5) rather than harsh commercial stripping solutions to preserve membrane integrity for reprobing.

What are the key considerations for using TNFRSF1A antibodies in flow cytometry experiments?

Successfully employing TNFRSF1A antibodies in flow cytometry requires attention to several technical aspects:

• Cell preparation: Single-cell suspensions must be prepared with minimal proteolytic damage to surface epitopes. Use EDTA-based cell dissociation solutions rather than trypsin when possible.

• Fixation impact: If fixation is necessary, use 2% paraformaldehyde rather than methanol, as the latter can disrupt membrane proteins and their associated epitopes.

• Titration optimization: Perform antibody titration experiments (typically testing 0.1-10 μg/mL) to determine the concentration yielding maximum signal-to-noise ratio .

• Biotin-streptavidin amplification: When using biotin-conjugated TNFRSF1A antibodies, secondary labeling with fluorophore-conjugated streptavidin (typically PE or APC) provides signal amplification, improving detection of low-abundance receptors.

• Compensation controls: For multicolor flow cytometry, establish proper compensation using single-stained controls to account for spectral overlap between biotin-streptavidin detection systems and other fluorophores.

• Receptor internalization: TNF receptor internalization occurs rapidly after ligand binding, so maintain cells at 4°C during staining procedures to minimize this effect.

• Validation using TNFRSF1A knockdown: Include cells with TNFRSF1A knockdown (via siRNA approaches) as negative controls to definitively establish staining specificity .

How can TNFRSF1A antibodies be employed in functional assays studying TNF signaling pathways?

TNFRSF1A antibodies serve as valuable tools for investigating TNF signaling mechanisms:

• Neutralization assays: Anti-TNFRSF1A antibodies can block TNF-α-induced cytotoxicity in susceptible cell lines like L-929 mouse fibroblasts. This approach allows dose-response studies measuring the antibody's neutralizing capacity (ND50) against recombinant TNF-α .

• Signaling pathway analysis: TNFRSF1A antibodies can be used to examine downstream signaling events by blocking receptor-ligand interaction. For example, pre-incubation of cells with anti-TNFRSF1A antibodies (4-10 μg/mL) inhibits TNF-α-induced phosphorylation of NF-κB subunits (p65, p105) in a dose-dependent manner .

• Cell proliferation and migration assays: Functional consequences of TNFRSF1A signaling can be assessed using cell counting kit-8 (CCK-8) assays and transwell migration assays after antibody treatment, as demonstrated in glioma cell lines .

• Gene expression modulation: TNFRSF1A antibodies can be used to examine how receptor blockade affects expression of downstream genes, such as stemness markers OCT-4 and NANOG in dental pulp stem cells .

• Receptor internalization studies: Biotinylated antibodies facilitate tracking of receptor internalization dynamics through time-course immunofluorescence studies combined with confocal microscopy.

What are the recommended approaches for troubleshooting non-specific binding with biotin-conjugated TNFRSF1A antibodies?

Non-specific binding issues can be addressed through systematic troubleshooting:

• Endogenous biotin blocking: Tissues and cells may contain endogenous biotin that can cause background signal. Pre-block samples with avidin followed by biotin before applying biotinylated antibodies.

• Secondary reagent optimization: When using streptavidin-conjugated detection reagents, titrate concentrations to minimize background while maintaining specific signal. Typically, 1:1000-1:5000 dilutions are appropriate.

• Alternative blocking reagents: If milk-based blocking solutions produce high background, switch to 2-3% BSA or commercial blocking reagents specifically designed for biotin-streptavidin systems.

• Cross-adsorbed streptavidin: Use cross-adsorbed streptavidin conjugates specifically tested for minimal binding to non-target elements in your experimental system.

• Stringent washing protocols: Implement more stringent washing procedures between incubation steps, using TBST with higher Tween-20 concentrations (0.1-0.5%) to reduce non-specific interactions.

• Epitope-specific validation: Compare results from different anti-TNFRSF1A antibodies targeting distinct epitopes (such as AA 20-43 versus AA 248-428) to distinguish between specific and non-specific signals .

How should I design experiments to compare TNFRSF1A expression levels across different cell types or tissue samples?

Designing robust comparative experiments requires standardized protocols and appropriate controls:

When comparing TNFRSF1A expression across samples, several precautions are essential. First, standardize sample collection and processing methods to minimize technical variability. For transcriptional analysis, the GAPDH primer sequences (forward: 5′-AGGTCGGAGTCAACGGATTT-3′, reverse: 5′-ATCTCGCTCCTGGAAGATGG-3′) have been validated for normalization purposes . For protein quantification, include gradient concentrations of recombinant TNFRSF1A protein to establish a standard curve for absolute quantification. Additionally, consider analyzing both membrane-bound and soluble forms of TNFRSF1A, as their ratio may have biological significance beyond total expression levels.

What controls should I include when using TNFRSF1A antibodies in neutralization experiments?

Neutralization experiments require rigorous controls to ensure valid interpretation:

• Isotype control antibody: Include a matched isotype control (Mouse IgG1 for monoclonal antibodies or non-immune rabbit IgG for polyclonal antibodies) at equivalent concentrations to distinguish specific neutralization from non-specific antibody effects .

• Dose-response assessment: Test a range of antibody concentrations (typically 0.1-50 μg/mL) to establish the neutralization dose-response curve and determine the ND50 (concentration yielding 50% inhibition) .

• Alternative receptor blockade: Include antibodies against TNF-RII/TNFRSF1B to distinguish receptor-specific effects from general TNF signaling inhibition.

• Cytotoxicity controls: Ensure the antibody itself does not induce cytotoxicity by performing viability assays with antibody alone (without TNF-α stimulation).

• Timing controls: Include time-course experiments to determine optimal pre-incubation times for receptor blockade (typically 30-60 minutes) before TNF-α challenge.

• Positive control inhibitors: Include established TNF-α inhibitors (such as etanercept or infliximab) as reference standards for comparison with anti-TNFRSF1A neutralization.

How do I interpret complex banding patterns in Western blots when using TNFRSF1A antibodies?

TNFRSF1A Western blots often present complex banding patterns requiring careful interpretation:

• Expected molecular weight: The primary TNFRSF1A band typically appears at approximately 55 kDa, representing the full-length receptor .

• Higher molecular weight bands: Bands above 100 kDa often represent receptor dimers or oligomers, particularly common in samples not fully reduced or when receptor clustering occurs following ligand binding.

• Lower molecular weight bands: Bands between 25-40 kDa may indicate proteolytic cleavage products of the receptor's extracellular domain, which occurs naturally during TNFRSF1A shedding .

• Glycosylation variants: Heterogeneous glycosylation can produce multiple bands or smears in the 50-60 kDa range, which can be verified by treating samples with glycosidases.

• Cross-reactivity assessment: To distinguish true TNFRSF1A variants from cross-reactive proteins, compare banding patterns across different anti-TNFRSF1A antibodies targeting distinct epitopes (such as AA 20-43 versus AA 248-428) .

• Validation with knockdown: siRNA-mediated knockdown should reduce intensity of all specific TNFRSF1A bands, as demonstrated in U251 and U87 cells using approaches targeting TNFRSF1A transcripts .

What are the methodological considerations for using TNFRSF1A antibodies in multiplex immunoassays?

Incorporating biotin-conjugated TNFRSF1A antibodies into multiplex platforms requires specialized considerations:

• Cross-reactivity matrix: Thoroughly test for cross-reactivity between the anti-TNFRSF1A antibody and other components in the multiplex panel. Documented absence of cross-reactivity with rhTGF-alpha, hTGF-beta 1, pTGF-beta 1, rhTGF-beta 1, pTGF-beta 1.2, pTGF-beta 2, recombinant chicken TGF-beta 3, rhTGF-beta 3, recombinant amphibian TGF-beta 5, rhTNF-beta, and rhTNF RII provides confidence in multiplex applications .

• Conjugation compatibility: When using biotin-conjugated antibodies, ensure no other biotin-streptavidin detection systems are employed within the same multiplex panel to prevent signal interference.

• Dynamic range optimization: Optimize antibody concentrations to ensure detection falls within the linear range of the assay, particularly important when target concentrations vary widely across different samples.

• Capture-detection antibody pairing: For sandwich assays, determine whether the biotin-conjugated antibody functions optimally as capture or detection reagent by testing both configurations.

• Bead-based considerations: For Luminex-type platforms, covalent coupling of antibodies to beads may provide more consistent results than biotin-streptavidin immobilization due to potential dissociation during extended incubations and wash steps.

• Multiplexing compatibility verification: Validate that anti-TNFRSF1A antibody performance remains consistent in single-plex versus multiplex formats by comparing standard curves for the target analyte under both conditions.

How can TNFRSF1A antibodies contribute to understanding TNF receptor shedding mechanisms?

TNFRSF1A antibodies provide valuable tools for investigating receptor shedding processes:

• Epitope-specific detection: Antibodies targeting different domains (extracellular versus intracellular) can selectively detect intact receptors versus shed ectodomains. The biotin-conjugated antibody targeting AA 20-43 (extracellular region) is particularly useful for monitoring shed receptor fragments .

• Shedding kinetics measurement: Time-course immunoprecipitation experiments using domain-specific antibodies can capture and quantify shed receptor fragments, providing insights into shedding dynamics under different stimuli.

• Metalloprotease inhibitor studies: Combining TNFRSF1A antibody detection with metalloprotease inhibitors (TAPI-1, TAPI-2) can help identify specific proteases responsible for receptor cleavage in different cell types.

• Differential detection strategies: Paired antibody approaches using membrane-proximal epitope (AA 20-43) and distal domain (AA 248-428) antibodies can distinguish between different cleavage products and shedding mechanisms .

• Functional consequences: Anti-TNFRSF1A neutralizing antibodies can help determine how receptor shedding affects TNF signaling by comparing cellular responses before and after shedding induction.

What strategies can improve reproducibility when using TNFRSF1A antibodies across different experimental systems?

Enhancing reproducibility requires systematic validation and standardization:

• Antibody validation metrics: Establish clear validation criteria including Western blot band patterns, knockdown efficiency requirements (>70% reduction), and cross-reactivity profiles before employing antibodies in complex systems .

• Lot-to-lot testing: Perform side-by-side comparisons between antibody lots using standardized positive control samples (like HL-60 cells) to calibrate working dilutions and detection parameters .

• Epitope sequence conservation: When working across species, verify epitope conservation through sequence alignment. The antibody targeting AA 20-43 shows documented cross-reactivity with human, mouse, rat, monkey, cow, dog and rabbit TNFRSF1A due to sequence conservation in this region .

• Reference sample repository: Maintain frozen aliquots of well-characterized positive control samples to use as standards across experiments and validate new antibody lots.

• Protocol standardization: Develop detailed standard operating procedures (SOPs) covering sample preparation, antibody dilution, incubation times/temperatures, and detection methods to minimize technical variability.

• Objective quantification methods: Implement digital image analysis algorithms for quantifying Western blot band intensity and immunostaining signals to reduce subjective assessment bias.

How can I apply TNFRSF1A antibodies to investigate therapeutic targeting of TNF signaling pathways in disease models?

TNFRSF1A antibodies enable several approaches for studying therapeutic interventions:

• Target validation: In disease models where TNF signaling is implicated (like glioma), TNFRSF1A knockdown experiments can validate the receptor as a therapeutic target by demonstrating effects on cell proliferation and migration .

• Mechanism distinction: Comparing effects of anti-TNF-α antibodies versus anti-TNFRSF1A antibodies can distinguish between ligand-targeting and receptor-targeting therapeutic approaches.

• Combination therapy assessment: TNFRSF1A neutralizing antibodies can be used alongside other pathway inhibitors to identify synergistic therapeutic combinations that more effectively block downstream signaling.

• Biomarker development: Flow cytometric quantification of TNFRSF1A expression using biotinylated antibodies can help identify patient populations likely to respond to TNF-pathway targeting therapies.

• Resistance mechanism elucidation: In models developing resistance to TNF-pathway inhibitors, TNFRSF1A antibodies can help identify receptor modifications, expression changes, or signaling adaptations contributing to therapeutic escape.

• In vivo imaging: Biotin-conjugated TNFRSF1A antibodies, when paired with imaging-compatible streptavidin conjugates, can facilitate non-invasive monitoring of receptor expression in preclinical disease models.