TNFAIP3 Antibody

What is TNFAIP3 and why is it important in immunological research?

TNFAIP3, commonly known as A20, is a ubiquitin-editing enzyme containing both ubiquitin ligase and deubiquitinase activities. It functions as a critical negative regulator of NF-κB signaling and plays important roles in inflammatory responses. TNFAIP3 is rapidly and transiently induced by TNF-α, inhibiting NF-κB-dependent gene expression and protecting cells from TNF-α-cytotoxicity . The protein is crucial for investigating:

• Inflammatory pathway regulation

• Immune response modulation

• Cell death mechanisms

• Various disease models including lymphomas and autoimmune disorders

TNFAIP3 is located on chromosome band 6q23, a region frequently deleted in B cell lymphomas, and has been identified as a tumor suppressor gene in Hodgkin lymphoma and several subtypes of non-Hodgkin lymphomas .

Selection should be based on:

• Target epitope: Different antibodies target various regions of TNFAIP3. For example, MAB75981 targets Lys91-Leu263 regions , while others may target C-terminal or N-terminal regions.

• Validated applications: Some antibodies perform better in specific applications. For instance, MAB7598 has been validated for Western blotting in HepG2 and NCI-H460 cell lines , while others show stronger performance in IHC or flow cytometry.

• Species reactivity: Confirm cross-reactivity with your experimental model. Most antibodies react with human TNFAIP3, but cross-reactivity with mouse or rat varies .

• Conjugation needs: Available options include unconjugated antibodies and those conjugated to fluorescent dyes like CoraLite 594 or FITC for direct detection .

• Clone validation data: Review immunoblot data, IHC images, and independent validation studies before selection .

What are the optimal fixation and retrieval methods for TNFAIP3 immunohistochemistry?

For successful TNFAIP3 immunohistochemistry:

• Fixation: Immersion fixation in paraformaldehyde or formalin is commonly used for tissue sections .

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using basic buffer (pH 9.0) is recommended for most TNFAIP3 antibodies, though citrate buffer (pH 6.0) may also be used as an alternative .

• Protocol example: For paraffin-embedded sections, incubate with primary antibody (e.g., MAB75981) at 1 μg/ml overnight at 4°C after heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic. Follow with appropriate HRP-DAB staining systems and hematoxylin counterstaining .

In immersion fixed paraffin-embedded sections of normal breast and liver tissues, specific TNFAIP3 staining localizes to cytoplasm and glandular cells or hepatocytes, respectively .

How can researchers validate TNFAIP3 antibody specificity?

Comprehensive validation should include:

• Positive and negative controls: Use cell lines with known TNFAIP3 expression (HepG2, Jurkat, HeLa as positive controls) .

• Western blot validation: Confirm the antibody detects a specific band at approximately 90-95 kDa under reducing conditions .

• Knockout/knockdown validation: Compare staining between wild-type and TNFAIP3-deficient samples.

• Cross-reactivity testing: Test reactivity against recombinant fragments of TNFAIP3. For example, MAB7598 showed no cross-reactivity with recombinant human A20/TNFAIP3 (aa 440-790) in direct ELISAs .

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm binding specificity.

What are the critical considerations when using TNFAIP3 antibodies in disease models?

When investigating TNFAIP3 in disease models:

• Expression dynamics: TNFAIP3 expression varies between relapse and remission states in disease models. For instance, in MOG-AAD patients, TNFAIP3 levels increase in CD4+ T cells during remission compared to relapse .

• Stimulation conditions: Consider the effects of antigen and drug stimulations on TNFAIP3 expression. In MOG-AAD patient samples, dexamethasone treatment increased TNFAIP3 expression compared to MOG antigen alone stimulation, with higher expression in non-relapse samples .

• Correlation with pathway components: TNFAIP3 expression shows negative correlation with NFκB subunits p50 and p65, reflecting its role in pathway regulation .

• Cell type-specific expression: TNFAIP3 regulation differs among cell types (CD4+ T cells, CD19+ B cells, CD14+ monocytes), with CD4+ T cells showing significant role in TNFAIP3 regulation in certain disease models .

• Time-course considerations: Expression can change rapidly after stimulation, necessitating careful time-point selection .

How can multiplex imaging be optimized when using TNFAIP3 antibodies?

For successful multiplex imaging with TNFAIP3 antibodies:

• Antibody selection: Choose conjugated antibodies like CoraLite 594-conjugated anti-TNFAIP3 (CL594-66695) or FITC-conjugated variants for direct fluorescence detection .

• Panel design: When combining with other markers:

  • Avoid spectral overlap between fluorophores

  • Consider sequential staining for multiple primary antibodies from the same species

  • Use appropriate negative and single-stain controls

• Optimization example: For TNFAIP3 subcellular localization in HL-60 cells, Mouse Anti-Human A20/TNFAIP3 (MAB75981) was used at 25 μg/mL for 3 hours at room temperature, followed by NorthernLights 557-conjugated secondary antibody and DAPI counterstaining, revealing specific cytoplasmic localization .

• Imaging parameters: Use appropriate exposure settings to detect the specific localization pattern (primarily cytoplasmic for TNFAIP3) .

What are the technical challenges in quantifying TNFAIP3 expression across different experimental systems?

Researchers face several challenges when quantifying TNFAIP3:

• Isoform diversity: At least two isoforms of TNFAIP3 exist, potentially complicating detection depending on the antibody epitope .

• Molecular weight variations: Observed molecular weight may differ from calculated weight (calculated: 90 kDa; observed: 80-95 kDa) depending on post-translational modifications and experimental conditions .

• Expression kinetics: TNFAIP3 is rapidly and transiently induced by TNF-α, requiring careful timing of experiments .

• Normalization strategies: For quantitative PCR, GAPDH has been successfully used as an endogenous control to normalize for RNA amount differences across samples .

• Detection sensitivity: Different methodologies show varying sensitivity:

  • Western blot using reducing conditions and Immunoblot Buffer Group 1 has been successful for detecting TNFAIP3 in HepG2 and NCI-H460 lysates

  • Simple Western™ can detect TNFAIP3 at approximately 101 kDa under reducing conditions using the 12-230 kDa separation system

  • qPCR with FAM-labeled primers provides sensitive detection of relative expression levels

How can TNFAIP3 antibodies be used to investigate NF-κB pathway dynamics?

To investigate NF-κB pathway dynamics using TNFAIP3 antibodies:

• Stimulation time course: Design experiments with multiple time points (4h, 8h, 16h, 24h) after stimulation to capture the dynamic relationship between TNFAIP3 and NFκB components .

• Co-immunoprecipitation: Use anti-TNFAIP3 antibodies for immunoprecipitation to identify interacting partners in the NF-κB pathway .

• Correlation analysis: Examine the negative correlation between TNFAIP3 expression and NFκB subunits p50 and p65 through simultaneous detection in the same samples .

• Dose-response experiments: Investigate how different concentrations of stimulants (e.g., MOG antigen at 1 μg/ml vs. 10 μg/ml) affect TNFAIP3 expression and corresponding NF-κB activity .

• Dual staining approaches: Combine TNFAIP3 antibodies with antibodies against NF-κB components to visualize their reciprocal relationship in single cells.

What methodological approaches can resolve contradicting data when working with TNFAIP3 antibodies?

When faced with contradictory results:

• Multiple antibody validation: Use different antibody clones targeting distinct epitopes of TNFAIP3. Compare results between monoclonal antibodies like clone 775928 (MAB75981) and clone 775912 (MAB7598) .

• Complementary techniques: Combine multiple detection methods:

  • Validate protein expression using both Western blot and immunostaining

  • Correlate protein detection with mRNA levels using qPCR

  • Confirm subcellular localization using both immunofluorescence and fractionation approaches

• Biological replicates: Analyze multiple patient/donor samples to account for biological variability, as demonstrated in MOG-AAD studies with multiple patient samples (n=7) with longitudinal sampling .

• Technical controls: Include appropriate positive controls (e.g., HeLa, HepG2, Jurkat cells) and negative controls (secondary antibody only, isotype controls) in each experiment .

• Orthogonal validation: For critical findings, validate results using genetic approaches (siRNA, CRISPR) to confirm antibody specificity.

How can TNFAIP3 antibodies be leveraged in biomarker development for autoimmune conditions?

Based on recent findings:

• Longitudinal patient monitoring: TNFAIP3 levels in CD4+ T cells increase during remission and decrease during relapse in MOG-AAD patients, suggesting potential as a disease activity biomarker .

• Cell type-specific analysis: Focus on CD4+ T cells, which show significant regulation of TNFAIP3 in autoimmune conditions compared to other immune cell types .

• Treatment response prediction: Monitor TNFAIP3 expression changes in response to treatments like mycophenolate mofetil and dexamethasone to identify potential predictive markers .

• Standardized detection protocols: Develop consistent protocols using validated antibodies and normalization strategies for clinical application:

  • qPCR using GAPDH normalization for mRNA detection

  • Flow cytometry with calibrated antibody concentrations for protein detection

  • Western blot with standardized lysate preparation and loading controls

• Correlation with clinical measures: Establish relationships between TNFAIP3 levels and clinical disease activity metrics for validation as a clinically useful biomarker .

What are the optimal approaches for studying TNFAIP3 post-translational modifications?

To investigate TNFAIP3 post-translational modifications:

• Antibody selection: Choose antibodies that recognize specific modifications or those that bind regardless of modification state.

• Specialized techniques:

  • Immunoprecipitation followed by mass spectrometry

  • Phospho-specific or ubiquitin-specific Western blotting

  • Use of deubiquitinase inhibitors or phosphatase inhibitors during sample preparation

• Functional analysis: Correlate observed modifications with TNFAIP3's dual ubiquitin ligase and deubiquitinase activities to understand functional implications.

• Visualization strategies: Use proximity ligation assays to visualize interactions between TNFAIP3 and its modifiers or substrates in situ.

How can TNFAIP3 antibodies contribute to understanding tumor microenvironment dynamics?

For tumor microenvironment studies:

• Multiplex tissue analysis: Combine TNFAIP3 antibodies with markers for various cell types in the tumor microenvironment to map expression patterns.

• Context-dependent expression: Investigate TNFAIP3 expression in:

  • Tumor cells (using antibodies validated in cancer cell lines like HepG2)

  • Tumor-infiltrating immune cells

  • Stromal components

• Therapeutic response correlation: Monitor changes in TNFAIP3 expression following immunotherapy or conventional cancer treatments.

• Prognostic value assessment: Correlate TNFAIP3 expression patterns with clinical outcomes given its role as a tumor suppressor gene in Hodgkin lymphoma and non-Hodgkin lymphomas .

What are the most common issues in TNFAIP3 immunostaining and how can they be resolved?

Common problems and solutions include:

For immunohistochemistry, a successful protocol includes:

• Heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic

• Primary antibody incubation at 1 μg/ml overnight at 4°C

• Detection using HRP-DAB Cell & Tissue Staining Kit

• Hematoxylin counterstaining

What advanced control strategies ensure robust TNFAIP3 antibody-based experiments?

To ensure experimental robustness:

• Gradient controls: Test a range of antibody concentrations to determine optimal signal-to-noise ratio (e.g., 1:1000-1:6000 for WB, 1:400-1:1600 for IF) .

• Specificity controls:

  • Pre-absorption with immunizing peptide

  • Isotype-matched control antibodies

  • Comparison across multiple antibody clones (e.g., clone 775928 vs. clone 775912)

• Biological validation:

  • Stimulation controls: Compare TNF-α stimulated versus unstimulated samples

  • Knockdown/knockout controls: Use siRNA or CRISPR to generate TNFAIP3-deficient samples

• Technical replicates: Perform at least three independent experiments to ensure reproducibility.

• Loading/normalization controls: Use appropriate housekeeping proteins for Western blots and GAPDH for qPCR normalization .

How might new TNFAIP3 antibody technologies advance single-cell analysis approaches?

Emerging opportunities include:

• Single-cell resolution imaging: Use highly specific TNFAIP3 antibodies in CyTOF or imaging mass cytometry to profile TNFAIP3 expression at single-cell resolution within complex tissues.

• In situ protein interaction analysis: Develop proximity ligation assays using TNFAIP3 antibodies to visualize protein-protein interactions in intact cells and tissues.

• Spatial transcriptomics integration: Combine TNFAIP3 protein detection with RNA expression analysis in spatial contexts to correlate protein levels with transcriptional states.

• Live-cell imaging approaches: Develop cell-permeable fluorescently-labeled TNFAIP3 antibody fragments to track dynamic expression changes in living cells.

• Microfluidic applications: Incorporate TNFAIP3 antibodies into microfluidic antibody capture assays for single-cell protein quantification alongside other parameters.

What are the emerging applications of TNFAIP3 antibodies in therapeutic development research?

In therapeutic research contexts:

• Target validation: Use TNFAIP3 antibodies to validate pathway modulation in drug discovery pipelines targeting inflammatory and autoimmune conditions.

• Patient stratification: Develop standardized TNFAIP3 detection protocols to identify patient subsets that might benefit from specific therapeutic approaches.

• Pharmacodynamic biomarkers: Monitor TNFAIP3 expression changes as pharmacodynamic markers for drugs targeting NF-κB pathways.

• Combined biomarker panels: Integrate TNFAIP3 with other inflammatory markers to create comprehensive pathway activation profiles for precision medicine approaches.

• Therapeutic antibody development: Use insights from diagnostic antibodies to develop therapeutic antibodies targeting TNFAIP3-related pathways.