TNFAIP8L2 Antibody

What is TNFAIP8L2 and what are its primary functions in the immune system?

TNFAIP8L2 (also known as TIPE2) acts as a negative regulator of innate and adaptive immunity by maintaining immune homeostasis. This protein serves several critical functions:

• Negatively regulates Toll-like receptor and T-cell receptor function

• Prevents hyperresponsiveness of the immune system

• Inhibits JUN/AP1 and NF-kappa-B activation pathways

• Promotes Fas-induced apoptosis

TNFAIP8L2 is primarily expressed in immune tissues and cells, with a calculated molecular weight of approximately 21 kDa, though it is typically observed at 18-20 kDa in Western blot applications .

What applications are recommended for TNFAIP8L2 antibodies in research?

TNFAIP8L2 antibodies have been validated for multiple applications with specific dilution recommendations:

All applications should be optimized for specific experimental conditions as performance may vary between antibody lots and experimental systems .

How should TNFAIP8L2 antibodies be stored and handled for optimal performance?

For maximum antibody stability and performance, follow these guidelines:

• Store concentrated antibody at -20°C for long-term storage (typically stable for 12 months from receipt)

• For frequent use, aliquot and store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this significantly reduces antibody activity

• Most formulations contain 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability

• When working with the antibody, keep it on ice and minimize exposure to room temperature

Proper storage is critical for maintaining antibody performance. According to manufacturer recommendations, some TNFAIP8L2 antibodies can maintain stability at 4°C for up to 6 months after reconstitution, but this varies by product .

What is the optimal sample preparation protocol for TNFAIP8L2 Western blotting?

For successful detection of TNFAIP8L2 in Western blot applications:

• Sample Preparation:

  • Prepare cell or tissue lysates using standard lysis buffer containing protease inhibitors

  • Load 25-30 μg of total protein per lane

• Electrophoresis and Transfer:

  • Use 12-15% SDS-PAGE gels for optimal resolution of the 18-20 kDa TNFAIP8L2 protein

  • Transfer to PVDF or nitrocellulose membranes (PVDF often provides better results)

• Blocking and Antibody Incubation:

  • Block with 3% non-fat dry milk in TBST for 1-2 hours at room temperature

  • Incubate with primary TNFAIP8L2 antibody at 1:1000-1:2000 dilution overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated secondary antibody at 1:10000 dilution for 1 hour

• Detection:

  • Develop using ECL detection system with exposure times ranging from 10-30 seconds

  • TNFAIP8L2 typically appears as a band at 18-20 kDa

Positive controls should include THP-1 cells, mouse thymus tissue, mouse spleen tissue, or rat spleen tissue, where TNFAIP8L2 expression has been well-documented .

What controls are essential when validating TNFAIP8L2 antibody specificity?

Proper validation of TNFAIP8L2 antibody specificity requires several controls:

Positive Controls:

• Cell lines: THP-1 cells, RAW 264.7 cells

• Tissues: Mouse spleen, mouse thymus, rat spleen, human lymphoma tissue

Negative Controls:

• TNFAIP8L2 knockout cell lines or tissues

• TNFAIP8L2 siRNA-treated samples

• Secondary antibody-only controls to assess non-specific binding

Blocking Peptide Validation:

• Pre-incubating the antibody with a specific blocking peptide containing the epitope recognized by the antibody

• Compare staining patterns with and without blocking peptide

• Significant reduction in signal indicates antibody specificity

Recent knockout (KO) validation approaches have emerged as the gold standard for antibody validation, as demonstrated in several recent TNFAIP8L2 studies .

How does TNFAIP8L2 regulate autophagy through the RAC1-MTORC1 signaling axis?

TNFAIP8L2 plays a sophisticated role in regulating autophagy through its interaction with the RAC1-MTORC1 signaling pathway:

Mechanism of Action:

• TNFAIP8L2 directly binds to and blocks RAC1 GTPase activity

• TNFAIP8L2 competes with MTOR for binding to the GTP-bound state of RAC1

• This competition negatively regulates MTORC1 activity

• Despite suppressing MTOR activity under glutamine and serum starvation, TNFAIP8L2 overexpression fails to induce autophagy flux

• TNFAIP8L2 appears to impair autophagic lysosome reformation (ALR) during prolonged starvation

• TNFAIP8L2 overexpression leads to defects in MTOR reactivation, disrupting autophagy flux and potentially leading to cell death

Experimental approaches for studying this interaction include:

• Co-Immunoprecipitation to detect TNFAIP8L2-RAC1 and RAC1-MTOR interactions

• Western blot analysis of LC3-II conversion and p62/SQSTM1 degradation

• Lysosomal morphology assessment using LAMP1 staining

TNFAIP8L2 deficiency has been shown to exacerbate inflammatory responses and lung injury by controlling MTOR activity in LPS-induced mouse models, highlighting the physiological relevance of this regulatory mechanism .

What methodological considerations are important when studying TNFAIP8L2's role in dendritic cell immune function?

When investigating TNFAIP8L2's role in dendritic cell (DC) immune function, consider these methodological approaches:

Experimental Design:

• Cell Models:

  • Primary bone marrow-derived dendritic cells (BMDCs)

  • DC cell lines (validate with primary cells)

• Genetic Manipulation:

  • TNFAIP8L2 knockout (KO) models: Show enhanced autophagy and improved immune response

  • TNFAIP8L2 overexpression (KI) models: Demonstrate inhibited autophagy and suppressed DC function

• Signaling Pathway Analysis:

  • Focus on the TAK1/JNK pathway, which is inhibited by TNFAIP8L2 in DCs

  • This inhibition results in downregulated autophagic activity

  • Western blot analysis of phosphorylated TAK1 and JNK provides insights into pathway activation

• Functional Assays:

  • DC maturation markers (CD80, CD86, MHC-II) by flow cytometry

  • Cytokine production profiles (IL-12, TNF-α, IL-6)

  • T cell stimulatory capacity through mixed lymphocyte reactions

• In Vivo Models:

  • LPS-induced endotoxemia reveals TNFAIP8L2's role in inflammation

  • Sepsis models provide physiologically relevant contexts

Research has demonstrated that TIPE2 can suppress the autophagic activity of DCs by inhibiting the TAK1/JNK signaling pathway, which negatively regulates the immune function of DCs during septic complications .

What are the technical challenges in optimizing immunohistochemistry protocols with TNFAIP8L2 antibodies?

Optimizing immunohistochemistry protocols for TNFAIP8L2 requires attention to several technical challenges:

• Antigen Retrieval Optimization:

  • Heat-induced epitope retrieval using TE buffer at pH 9.0 is recommended

  • Alternative: citrate buffer at pH 6.0

  • Insufficient retrieval is a common cause of weak staining

• Antibody Titration:

  • Start with the manufacturer's recommended range (typically 1:50-1:500)

  • Perform systematic titration experiments to determine optimal concentration

  • Different lots may require re-optimization

• Detection System Selection:

  • Polymer-based detection systems generally provide superior sensitivity

  • DAB (3,3'-diaminobenzidine) as chromogen for brightfield microscopy

  • For fluorescence detection, Alexa Fluor-conjugated secondary antibodies

• Tissue-Specific Considerations:

  • Recommended positive controls: human lymphoma tissue, human ovary tumor tissue, mouse spleen tissue

  • Fixation time affects epitope accessibility

  • Background staining can vary between tissue types

• Troubleshooting Common Issues:

  • High background: Increase blocking time/concentration or antibody dilution

  • Weak signal: Decrease antibody dilution, extend incubation time, or enhance antigen retrieval

  • Non-specific binding: Increase washing steps or add 0.1% Triton X-100 to wash buffer

Validation data indicates that mouse spleen tissue sections show robust and specific staining when processed with appropriate retrieval methods and antibody concentrations .

How can researchers design effective TNFAIP8L2 knockout/knockdown models for studying inflammatory responses?

Creating effective TNFAIP8L2 knockout/knockdown models requires systematic approaches:

• Knockout Strategy Options:

  • CRISPR/Cas9: Target early exons (exon 1 or 2) for complete disruption

  • Traditional homologous recombination: Replace critical exons with selection markers

  • Conditional knockout: Use Cre-loxP system for tissue-specific deletion

• Knockdown Approaches:

  • siRNA: Design multiple siRNAs targeting different regions of TNFAIP8L2 mRNA

  • shRNA: For stable knockdown, use lentiviral vectors with selection markers

  • Consider knockdown efficiency and potential for off-target effects

• Validation Methods:

  • Genomic verification: PCR and sequencing to confirm modifications

  • Transcript analysis: RT-qPCR to quantify TNFAIP8L2 mRNA levels

  • Protein verification: Western blot using validated antibodies

  • Functional validation: Assess known TNFAIP8L2-dependent pathways

• Inflammatory Response Assessment:

  • Compare baseline vs. stimulated conditions (LPS, TNF-α, IL-1β)

  • Measure cytokine production (ELISA, multiplex assays)

  • Analyze signaling pathway activation (phospho-specific Western blots)

  • Assess cell-specific responses in different immune cell populations

Studies have demonstrated that TNFAIP8L2 knockout in dendritic cells significantly enhances autophagy and improves immune responses in sepsis models, providing valuable insights into the protein's role in regulating inflammatory responses .

What are the most effective approaches for investigating protein-protein interactions involving TNFAIP8L2?

To effectively study TNFAIP8L2 protein interactions:

• Co-Immunoprecipitation (Co-IP):

  • Use antibodies specifically validated for immunoprecipitation (IP)

  • Optimize lysis conditions to preserve interactions (mild, non-denaturing buffers)

  • Include crucial controls:

    • Input controls (5-10% pre-IP lysate)

    • Negative controls (IgG isotype, TNFAIP8L2 knockout samples)

    • Reciprocal IP to confirm interactions

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein interactions in situ

  • Particularly useful for examining TNFAIP8L2 interactions with signaling molecules

  • Provides spatial information about interaction locations within cells

• Competitive Binding Assays:

  • For studying TNFAIP8L2's competition with MTOR for RAC1 binding

  • Express increasing amounts of TNFAIP8L2 to assess displacement of MTOR

  • Quantify relative amounts of binding partners

• Pull-down Assays:

  • Use purified recombinant TNFAIP8L2 as bait

  • GST-tagged or His-tagged constructs allow for efficient purification

  • Can identify direct vs. indirect interactions

• Nucleotide State Manipulation:

  • For GTPase interactions (e.g., RAC1), control nucleotide binding state

  • Use GTPγS (non-hydrolyzable GTP analog) for GTP-bound state

  • Use GDP for GDP-bound state

  • Include magnesium in buffers to stabilize nucleotide binding

Research has demonstrated that TNFAIP8L2 can directly bind to and block RAC1 GTPase activity, and it can compete with MTOR for binding to the GTP-bound state of RAC1, providing critical insights into its regulatory functions .

How can researchers troubleshoot inconsistent results when using TNFAIP8L2 antibodies?

When encountering inconsistent results with TNFAIP8L2 antibodies, implement this systematic troubleshooting approach:

• Antibody Validation:

  • Verify antibody specificity using TNFAIP8L2 knockout/knockdown controls

  • Test multiple antibodies targeting different epitopes

  • Check lot-to-lot consistency if using different batches

• Sample Preparation Optimization:

  • Ensure complete protein denaturation for Western blot

  • Verify sample integrity (minimal protein degradation)

  • Consider post-translational modifications that might affect epitope recognition

  • Standardize lysate preparation protocols

• Protocol Adjustments:

  • Western Blot:

    • Adjust protein loading (25-30 μg typically optimal)

    • Optimize gel percentage (12-15% recommended for 18-20 kDa TNFAIP8L2)

    • Try different transfer conditions and membrane types

  • Immunohistochemistry/Immunofluorescence:

    • Test different antigen retrieval methods

    • Adjust antibody concentration and incubation time

    • Modify blocking conditions to reduce background

• Technical Variables to Control:

  • Buffer composition (pH, salt concentration, detergents)

  • Incubation temperature and duration

  • Secondary antibody selection and dilution

  • Detection system sensitivity

• Tissue/Cell-Specific Considerations:

  • Expression levels vary across tissues (highest in immune tissues)

  • Cell activation status affects expression

  • Species differences in epitope sequences may affect cross-reactivity

Research data indicates that TNFAIP8L2 is consistently detected at 18-20 kDa in Western blot applications, despite its calculated molecular weight of 21 kDa, which could be due to post-translational modifications or cleavage .

What are the key methodological approaches for studying TNFAIP8L2's role in autophagy regulation?

To effectively investigate TNFAIP8L2's role in autophagy regulation:

• Autophagy Flux Assessment:

  • LC3-II conversion: Western blot analysis of LC3-I to LC3-II conversion

  • p62/SQSTM1 degradation: Monitor levels as marker of autophagy completion

  • Tandem-tagged LC3 (mRFP-GFP-LC3): Distinguish autophagosomes from autolysosomes

  • Include bafilomycin A1 treatment to block autophagosome-lysosome fusion

• Genetic Manipulation Approaches:

  • TNFAIP8L2 overexpression: Assess effects on autophagy markers

  • TNFAIP8L2 knockdown/knockout: Determine if autophagy is enhanced

  • Rescue experiments: Re-express TNFAIP8L2 in knockout models

• Signaling Pathway Analysis:

  • MTORC1 activity: Monitor phosphorylation of S6K and 4E-BP1

  • RAC1 activity: GTP-bound RAC1 pull-down assays

  • TAK1/JNK pathway: Assess phosphorylation status by Western blot

  • Competition assays: Evaluate TNFAIP8L2 vs. MTOR for RAC1 binding

• Autophagic Lysosome Reformation (ALR) Assessment:

  • LAMP1 staining: Monitor lysosomal morphology and distribution

  • Lysosomal pH measurements: LysoTracker or LysoSensor dyes

  • Live-cell imaging: Track dynamics of autophagosome-lysosome fusion

• Physiological Context:

  • Nutrient starvation responses: Serum and glutamine deprivation

  • Inflammatory stimuli: LPS, TNF-α treatment

  • In vivo models: LPS-induced endotoxemia, sepsis models

Research has demonstrated that TNFAIP8L2 overexpression fails to induce autophagy flux despite suppressing MTOR activity under starvation conditions. Instead, it appears to impair autophagic lysosome reformation during prolonged starvation, highlighting its complex regulatory role in autophagy .