TNFAIP8L2 Antibody, HRP conjugated

What is TNFAIP8L2/TIPE2 and why is it significant in immunological research?

TNFAIP8L2 (tumor necrosis factor alpha-induced protein 8-like protein 2), also known as TIPE2, is a 184 amino acid protein that belongs to the TNFAIP8 family. It plays a crucial role as a negative regulator of both innate and adaptive immunity by maintaining immune homeostasis. The significance of TIPE2 lies in its ability to prevent hyperresponsiveness of the immune system by negatively regulating Toll-like receptor (TLR) and T-cell receptor (TCR) functions. It inhibits JUN/AP1 and NF-kappa-B activation pathways and promotes Fas-induced apoptosis .

TIPE2 is predominantly expressed in lymphoid tissues including spleen, thymus, small intestine, and lymph nodes, with lower expression levels in colon, lung, and skin. The gene encoding TIPE2 maps to human chromosome 1, which contains over 3,000 genes and comprises nearly 8% of the human genome .

What are the key tissue expression patterns and cellular localization of TNFAIP8L2?

TNFAIP8L2/TIPE2 exhibits a distinct tissue expression pattern primarily in immune-related tissues:

At the cellular level, TIPE2 is predicted to be active in the cytoplasm where it exerts its function as a negative regulator of T cell activation and inflammatory responses . Immunofluorescence studies using anti-TNFAIP8L2 antibodies have been instrumental in confirming this localization pattern, with recommended dilutions of 1:50-400 for optimal visualization .

What are the optimal applications for TNFAIP8L2 Antibody, HRP conjugated?

The HRP-conjugated TNFAIP8L2 antibody is versatile and can be used in multiple experimental applications. Based on validated protocols, the following applications and their recommended dilutions have been established:

The antibody has been validated to react with human TNFAIP8L2 with predicted cross-reactivity to mouse, rat, dog, cow, pig, horse, and rabbit proteins, making it suitable for comparative studies across multiple species .

How should I optimize Western blot protocols when using TNFAIP8L2 Antibody, HRP conjugated?

For Western blot optimization with HRP-conjugated TNFAIP8L2 antibody:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for protein extraction

  • Load 20-50 μg of total protein per lane

  • Include positive controls such as lysates from mouse thymus, spleen, or small intestine

• Membrane transfer and blocking:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Start with 1:1000 dilution for initial optimization

  • Incubate overnight at 4°C

  • Since the antibody is directly HRP-conjugated, no secondary antibody is needed, which reduces background and cross-reactivity issues

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Expected molecular weight of TNFAIP8L2 is approximately 21 kDa

  • Exposure time varies based on expression level: 10 seconds to 5 minutes

• Troubleshooting:

  • If signal is weak, increase antibody concentration (up to 1:300)

  • If background is high, increase washing steps or dilute antibody further (up to 1:5000)

How can TNFAIP8L2 Antibody, HRP conjugated be used to investigate autophagy mechanisms?

Recent research has revealed TNFAIP8L2/TIPE2's role in autophagy regulation, making HRP-conjugated antibodies valuable tools for investigating this process. TIPE2 competes with MTOR for binding to the GTP-bound state of RAC1 and negatively regulates MTORC1 activity .

To investigate TIPE2's role in autophagy:

• Co-immunoprecipitation experiments:

  • Use the HRP-conjugated antibody at 1:50 dilution for immunoprecipitation

  • Investigate interactions between TIPE2, RAC1, and MTOR

  • Analyze whether TIPE2 competes with MTOR for RAC1 binding

• Autophagy flux analysis:

  • Examine LC3-II and p62 levels in cells with varying TIPE2 expression

  • The HRP-conjugated antibody can be used at 1:1000 for Western blot detection

  • Compare autophagy markers under normal and starvation conditions

• Autophagic lysosome reformation (ALR) assessment:

  • TIPE2 specifically impairs ALR during prolonged starvation

  • Use the antibody to correlate TIPE2 expression with lysosomal tubulation and reformation events

Research has shown that TIPE2 overexpression leads to defects in MTOR reactivation and disrupts autophagy flux, which can lead to cell death. This suggests that TIPE2 could be a therapeutic target for disorders caused by defective autophagy, including neurodegeneration, cancer, heart disease, and infectious diseases .

What methods can be used to study TNFAIP8L2's role in Type 2 Diabetes Mellitus (T2DM)?

TNFAIP8L2/TIPE2 has been implicated in Type 2 Diabetes Mellitus (T2DM), with significant upregulation observed in peripheral blood mononuclear cells (PBMCs) from T2DM patients . The HRP-conjugated antibody can facilitate several experimental approaches:

• Expression analysis in diabetic models:

  • Western blot analysis of TIPE2 expression in PBMCs from T2DM patients vs. healthy controls

  • Recommended dilution: 1:1000

  • Expected finding: Significant increase in TIPE2 expression in T2DM patients

• Correlation studies with inflammatory markers:

  • Research has established negative correlations between TIPE2 mRNA expression and serum levels of:

    • TNF-α (r = -0.3353, P = 0.01)

    • IL-6 (r = -0.267, P = 0.0427)

    • hsCRP (r = -0.2982, P = 0.02)

  • Use combined ELISA and Western blot approaches to validate these relationships

• High glucose treatment experiments:

  • Treat differentiated THP-1 cells with high glucose (50 mmol/L)

  • Monitor TIPE2 expression changes using the HRP-conjugated antibody

  • Expected result: Upregulation of TIPE2 and increased cytokine secretion

• Functional studies:

  • Use siRNA to knock down TIPE2 (combined with antibody detection)

  • Overexpress TIPE2 using adenoviral vectors

  • Measure TNF-α and IL-6 concentrations in the cell culture supernatants

  • Expected findings: siTIPE2 exacerbates TNF-α and IL-6 production under high glucose, while TIPE2 overexpression reverses increased TNF-α

What are the optimal storage and handling conditions for TNFAIP8L2 Antibody, HRP conjugated?

To maintain antibody functionality and prevent degradation:

• Storage temperature:

  • Store at -20°C for long-term preservation

  • Avoid repeated freeze-thaw cycles by aliquoting into multiple vials

• Buffer composition:

  • Typically provided in aqueous buffered solution containing:

    • 0.01M TBS (pH 7.4)

    • 1% BSA

    • 0.02% Proclin300 (preservative)

    • 50% Glycerol (stabilizer)

• Stability:

  • Long-term stability: 12 months from date of receipt at -20°C

  • After thawing: 6 months at 2-8°C

• Handling precautions:

  • Let the antibody reach room temperature before opening

  • Centrifuge briefly before use to collect contents at the bottom of the tube

  • Avoid vortexing to prevent protein denaturation

  • Return to storage promptly after use

How can I validate the specificity of the TNFAIP8L2 Antibody, HRP conjugated in my experimental system?

To ensure antibody specificity and reliability in your experimental system:

• Positive controls:

  • Use tissues with known high TIPE2 expression:

    • Mouse thymus

    • Mouse spleen

    • Mouse small intestine

  • Human PBMC samples, particularly from T2DM patients

• Negative controls:

  • Isotype control antibodies

  • TIPE2 knockout or knockdown samples

  • Pre-absorption with immunizing peptide (if available)

• Cross-reactivity assessment:

  • Test against related proteins in the TNFAIP8 family

  • Verify staining patterns across multiple species if working with non-human models

• Multiple detection methods:

  • Compare results across different applications (WB, IHC, IF)

  • Look for consistent molecular weight (21 kDa) and expression patterns

• Antibody validation experiments:

  • siRNA knockdown followed by Western blot

  • Overexpression systems followed by detection

  • Co-localization with other markers in IF or IHC

How might TNFAIP8L2 antibodies contribute to understanding the protein's role in inflammatory diseases beyond T2DM?

TNFAIP8L2/TIPE2 has been implicated in multiple inflammatory conditions. Research using HRP-conjugated antibodies can advance our understanding of its role in:

• Systemic lupus erythematosus (SLE):

  • TNFAIP8L2 downregulation is associated with systemic autoimmunity

  • Research questions to explore:

    • How does TIPE2 expression correlate with disease activity?

    • Can TIPE2 levels predict treatment response?

• Hepatitis B:

  • Abnormal TIPE2 expression has been observed in PBMCs from chronic hepatitis B patients

  • Research approaches:

    • Immunohistochemistry of liver biopsies using dilutions of 1:200-400

    • Correlation of viral load with TIPE2 expression levels

• Asthma:

  • TIPE2 expression abnormalities reported in asthmatic children

  • Potential investigations:

    • Immunohistochemical analysis of bronchial biopsies

    • Western blot analysis of TIPE2 expression in response to allergen challenge

• Tumor progression:

  • TIPE2 can function as a biomarker and inhibitor of tumor progression by targeting the RAS signaling pathway

  • Research applications:

    • IHC analysis of tumor tissues at 1:200-400 dilution

    • Correlation of TIPE2 expression with tumor stage and prognosis

How can researchers integrate TNFAIP8L2 antibody-based approaches with emerging technologies to advance understanding of immune homeostasis?

Integrating TNFAIP8L2 antibody techniques with cutting-edge technologies:

• Single-cell analysis:

  • Combine antibody-based detection with single-cell RNA sequencing

  • Identify cell populations with differential TIPE2 expression

  • Correlate with immune cell activation states

• Proximity ligation assays:

  • Use HRP-conjugated TNFAIP8L2 antibodies in combination with antibodies against:

    • RAC1 to study interaction dynamics

    • MTOR to investigate competition mechanisms

    • Phosphoinositide species to explore TIPE2's role in leukocyte polarity

• CRISPR-Cas9 genome editing:

  • Generate TIPE2 knockout or point mutant cell lines

  • Use the HRP-conjugated antibody to validate editing efficiency

  • Study functional consequences on immune signaling pathways

• Bioinformatic approaches:

  • Integrate antibody-based protein quantification with transcriptomic data

  • Develop predictive models for TIPE2-related disease progression

  • Identify novel TIPE2 interaction partners through network analysis

• Therapeutic development:

  • Screen for small molecules that modulate TIPE2 expression or function

  • Use the HRP-conjugated antibody to measure intervention efficacy

  • Investigate TIPE2 as a biomarker for treatment response