TNFAIP8L2 Antibody, Biotin 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, serves as a critical negative regulator of innate and adaptive immunity that plays a fundamental role in maintaining immune homeostasis . This 184 amino acid protein is preferentially expressed in myeloid cell types, with highest expression in spleen, thymus, small intestine, and lymph node, and lower levels in colon, lung, and skin . TNFAIP8L2 functions as a molecular "brake" for immunometabolism, with expression that drastically decreases in lipopolysaccharide (LPS)-stimulated macrophages .

Research has demonstrated that TNFAIP8L2 deficiency leads to heightened expression of genes enriched for leukocyte activation and lipid biosynthesis pathways, while also affecting mitochondrial respiration rates in macrophages . The protein's unique anti-inflammatory and metabolic-modulatory functions make it a promising therapeutic target for cardiovascular diseases and cancer research .

What applications can biotin-conjugated TNFAIP8L2 antibody be used for in experimental research?

The biotin-conjugated TNFAIP8L2 polyclonal antibody demonstrates versatility across multiple research applications:

• Western Blotting (WB): Effective at dilution ranges of 1:300-5000

• Enzyme-Linked Immunosorbent Assay (ELISA): Optimal at dilutions of 1:500-1000

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Recommended at 1:200-400 dilutions

• Immunohistochemistry on frozen tissues (IHC-F): Best results at 1:100-500 dilutions

The biotin conjugation provides significant advantages through compatibility with streptavidin-based detection systems, offering enhanced sensitivity and signal amplification compared to conventional antibody detection methods. This facilitates both chromogenic and fluorescent visualization techniques depending on the experimental requirements.

What are the optimal storage conditions for maintaining antibody activity?

For maximum stability and performance of the biotin-conjugated TNFAIP8L2 antibody:

• Store the antibody at -20°C for long-term preservation (up to 12 months)

• The provided storage buffer contains 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol, which protects antibody integrity during freeze-thaw cycles

• For working solutions, maintain at 4°C for short-term use (up to one week)

• Minimize repeated freeze-thaw cycles which can degrade antibody performance

• Centrifuge briefly before opening to collect solution at the bottom of the vial

How should experimental controls be designed when using TNFAIP8L2 antibody in cellular studies?

Designing appropriate controls is critical when working with TNFAIP8L2 antibody:

• Positive tissue controls:

  • Include spleen or lymph node sections (high TNFAIP8L2 expression) for IHC applications

  • Use isolated myeloid cells (preferably unstimulated) for Western blot and flow cytometry

• Negative controls:

  • Utilize TNFAIP8L2 knockdown or knockout cell lines/tissues

  • Validated siRNA sequences that have proven efficacy include:

    • siRNA1: 5ʹ-CAGGAAGCTGCTAGACGAA-3ʹ

    • siRNA2: 5ʹ-GCCACGTGTTTGATCACTT-3ʹ

    • siRNA3: 5ʹ-GCTGCTAGAGTTGGTGGAA-3ʹ

• Time-course considerations:

  • TNFAIP8L2 expression significantly decreases following LPS stimulation (>2-fold reduction after 1 hour)

  • Include both unstimulated and time-course stimulated samples when studying inflammatory responses

  • Western blot confirmation has validated this downregulation in bone marrow-derived macrophages

• Antibody controls:

  • Include secondary-only controls when using detection systems

  • For antigen competition assays, pre-incubate with immunizing peptide (derived from region 10-110/184 of human TNFAIP8L2)

How can researchers analyze contradictory data regarding TNFAIP8L2's role in autophagy regulation?

When encountering conflicting results in TNFAIP8L2 autophagy studies, consider these methodological approaches:

• Context-dependent molecular interactions:

  • TNFAIP8L2 competes with MTOR for binding to the GTP-bound state of RAC1, negatively regulating MTORC1 activity

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

  • Validate both MTOR inhibition (via phospho-S6K/4E-BP1) and autophagy markers (LC3-II, p62) simultaneously

• Expression level considerations:

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

  • Compare gain-of-function (overexpression) and loss-of-function (knockout/knockdown) approaches in parallel experiments

  • Titrate expression levels to determine threshold effects

• Temporal dynamics analysis:

  • TNFAIP8L2 specifically impairs autophagic lysosome reformation (ALR) during prolonged starvation rather than initial autophagosome formation

  • Design time-course experiments that distinguish between early autophagy induction and late-stage ALR processes

  • Monitor dynamic changes in both MTOR signaling and autophagic flux markers

• Structural and functional mutation studies:

  • Utilize TNFAIP8L2 K15,16Q mutant (decreased RAC1 binding ability) as a control

  • Compare wild-type TNFAIP8L2 effects to this binding-deficient mutant

  • Monitor the competition between TNFAIP8L2 and MTOR for RAC1 binding using co-immunoprecipitation approaches

What strategies can optimize Western blot protocols for TNFAIP8L2 detection in primary immune cells?

For optimal Western blot detection of TNFAIP8L2 in primary immune cells:

• Sample preparation:

  • For macrophages and myeloid cells, use RIPA buffer supplemented with protease and phosphatase inhibitors

  • Consider that TNFAIP8L2 expression decreases significantly after LPS stimulation (>2-fold reduction)

  • Include time-matched controls when working with stimulated cells

• Gel electrophoresis parameters:

  • Use 12-15% SDS-PAGE gels for optimal resolution of TNFAIP8L2 (~21 kDa)

  • Load 20-40 μg of total protein per lane from primary cell lysates

  • Include molecular weight markers that clearly indicate the 20-25 kDa range

• Transfer and detection optimization:

  • Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

  • Block with 5% BSA in TBST for 1 hour at room temperature

  • Incubate with biotin-conjugated TNFAIP8L2 antibody at 1:1000 dilution overnight at 4°C

  • Detect using streptavidin-HRP at 1:5000 for 1 hour at room temperature

• Signal development:

  • Use ECL substrate with appropriate sensitivity for expected expression levels

  • For weak signals, consider enhanced chemiluminescent substrates or longer exposure times

  • Validate bands by comparing with positive control tissues (spleen/lymphoid tissues)

How does TNFAIP8L2 regulate the RAC1-MTORC1 signaling axis?

TNFAIP8L2 exhibits complex regulatory functions in the RAC1-MTORC1 signaling pathway:

• Competitive binding mechanism:

  • TNFAIP8L2 directly binds to and blocks RAC1 GTPase activity, particularly interacting with GTP-bound RAC1

  • TNFAIP8L2 competes with MTOR for binding to RAC1, progressively abrogating RAC1-MTOR association in a dose-dependent manner

  • The TNFAIP8L2 K15,16Q mutation decreases its binding ability with RAC1, promoting RAC1-MTOR interaction when expressed

• Effect on MTORC1 activity:

  • Through competitive binding to RAC1, TNFAIP8L2 negatively regulates MTORC1 activity

  • Despite MTOR suppression, TNFAIP8L2 overexpression fails to induce autophagy flux under starvation conditions

  • This creates a unique situation where MTOR inhibition does not lead to expected autophagy induction

• Autophagy regulation:

  • TNFAIP8L2 specifically impairs autophagic lysosome reformation (ALR) during prolonged starvation

  • TNFAIP8L2 overexpression leads to defects in MTOR reactivation and disrupts autophagy flux

  • This disruption can ultimately lead to cell death under certain conditions

• Inflammatory response modulation:

  • TNFAIP8L2 deficiency exacerbates inflammatory responses and lung injury by affecting MTOR activity

  • This has been demonstrated in LPS-induced mouse endotoxemia models

  • The protein acts as a molecular "brake" for both innate and adaptive immunity

What methodologies best demonstrate TNFAIP8L2's effects on immune cell metabolism?

To effectively investigate TNFAIP8L2's impact on immune cell metabolism:

• Mitochondrial respiration analysis:

  • Utilize Seahorse metabolic analyzer to measure oxygen consumption rate (OCR) in cells with varied TNFAIP8L2 expression

  • Compare basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity

  • Research has demonstrated increased mitochondrial respiration rates in TNFAIP8L2-deficient macrophages

• Gene expression profiling approaches:

  • Perform microarray or RNA-seq analysis of cells with TNFAIP8L2 knockout or overexpression

  • Focus on genes enriched in leukocyte activation and lipid biosynthesis pathways

  • Use Gene Set Enrichment Analysis (GSEA) to identify affected pathways (e.g., "interferon signaling" and "cholesterol biosynthesis")

• Lipid metabolism assessment:

  • Measure cellular lipid content using fluorescent lipid dyes or mass spectrometry

  • Analyze expression of lipid biosynthesis enzymes via qPCR and Western blotting

  • Compare results between wild-type and TNFAIP8L2-deficient cells, with and without inflammatory stimulation

• Inflammation-metabolism intersection:

  • Design experiments that simultaneously track inflammatory responses and metabolic parameters

  • Include time course studies that capture the dynamic relationship between these processes

  • Correlate TNFAIP8L2 expression levels with both immune activation markers and metabolic readouts

How can researchers effectively investigate TNFAIP8L2's role in inflammatory disease models?

For robust investigation of TNFAIP8L2 in inflammatory disease contexts:

• In vivo disease model approaches:

  • Utilize TNFAIP8L2 knockout mice in models of inflammatory diseases (e.g., LPS-induced endotoxemia)

  • Compare inflammatory responses, tissue damage, and survival outcomes

  • Research has shown that TNFAIP8L2 deficiency exacerbates inflammatory responses and lung injury

• Cell-specific expression analysis:

  • Use the biotin-conjugated TNFAIP8L2 antibody for IHC-P (1:200-400) or IHC-F (1:100-500) in disease tissues

  • Compare expression patterns between healthy and diseased tissues

  • Correlate with markers of inflammation, tissue damage, and disease severity

• Molecular intervention strategies:

  • Test therapeutic approaches that target the RAC1-MTORC1-TNFAIP8L2 axis

  • Monitor changes in both inflammatory parameters and metabolic readouts

  • Investigate whether restoring normal TNFAIP8L2 expression or function ameliorates disease

• Translation to human disease:

  • Analyze TNFAIP8L2 expression in human inflammatory disease samples

  • Correlate expression levels with clinical outcomes and biomarkers

  • Use findings to assess potential as a therapeutic target for cardiovascular diseases and cancer

What are the key considerations for immunoprecipitation experiments using biotin-conjugated TNFAIP8L2 antibody?

For successful immunoprecipitation with biotin-conjugated TNFAIP8L2 antibody:

• Capture system optimization:

  • The biotin conjugation enables direct pull-down using streptavidin-coated beads

  • Use 2-5 μg antibody per 500 μg-1 mg of total protein lysate

  • Pre-clear lysates with unconjugated beads to reduce non-specific binding

• Buffer considerations:

  • Use mild lysis buffers (e.g., NP-40 or CHAPS-based) to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to prevent degradation

  • For detecting RAC1-TNFAIP8L2 interactions, supplement buffer with GTP or non-hydrolyzable GTP analogs

• Experimental controls:

  • Include IgG control for non-specific binding

  • Use TNFAIP8L2 K15,16Q mutant as a negative control for RAC1 interaction studies

  • Consider including both wild-type and constitutively active RAC1 (Q61L) for interaction studies

• Detection strategies:

  • For co-immunoprecipitation studies, blot for interacting partners (RAC1, MTOR)

  • Be aware that TNFAIP8L2 competes with MTOR for binding to RAC1 in a dose-dependent manner

  • When using cell lysates from stimulated cells, remember that TNFAIP8L2 expression decreases after LPS stimulation

How can researchers troubleshoot weak or non-specific signals in immunohistochemistry?

To resolve common issues in IHC applications:

• Antigen retrieval optimization:

  • Test multiple retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • For TNFAIP8L2, heat-induced epitope retrieval with citrate buffer is typically effective

  • Extend retrieval time (15-30 minutes) or increase temperature if signal is weak

• Antibody concentration and incubation:

  • For weak signals, increase antibody concentration (try 1:100 instead of 1:400)

  • Extend primary antibody incubation to overnight at 4°C

  • Ensure sections remain hydrated throughout the staining procedure

• Detection system enhancement:

  • Use streptavidin-HRP polymer systems for signal amplification

  • Consider biotin blocking when working with tissues that have endogenous biotin

  • For fluorescent detection, use streptavidin conjugated to bright, photostable fluorophores

• Tissue-specific considerations:

  • TNFAIP8L2 is most highly expressed in spleen, thymus, small intestine, and lymph node

  • Include these tissues as positive controls when optimizing protocols

  • For tissues with expected low expression, consider using more sensitive detection methods

What are the critical parameters for optimal flow cytometry with TNFAIP8L2 antibody?

For effective flow cytometry applications:

• Cell preparation:

  • For intracellular staining, use a gentle fixation protocol (2% paraformaldehyde for 10-15 minutes)

  • Permeabilize with 0.1% saponin or a commercial permeabilization buffer

  • Maintain cells at 4°C throughout the staining procedure to minimize antibody internalization

• Staining protocol:

  • Block Fc receptors before antibody addition to reduce background

  • Use the biotin-conjugated TNFAIP8L2 antibody at 1:50-200 dilution

  • Counterstain with streptavidin conjugated to bright fluorophores (PE, APC, or their tandems)

• Gating strategy design:

  • First gate on viable cells using appropriate viability dyes

  • For myeloid populations, use markers like CD11b, CD14, or F4/80

  • TNFAIP8L2 is preferentially expressed in myeloid cell types

• Controls and validation:

  • Include fluorescence minus one (FMO) controls

  • Use TNFAIP8L2 knockout or knockdown cells as negative controls

  • For stimulation experiments, include unstimulated controls (TNFAIP8L2 expression decreases after LPS stimulation)

What experimental approaches can identify novel TNFAIP8L2 interacting partners?

To discover new TNFAIP8L2 protein interactions:

• Proximity labeling techniques:

  • Fuse TNFAIP8L2 with BioID or APEX2 enzymes

  • These enzymes biotinylate proteins in close proximity

  • Capture biotinylated proteins using streptavidin and identify by mass spectrometry

  • Compare results with TNFAIP8L2 K15,16Q mutant to distinguish RAC1-dependent interactions

• Co-immunoprecipitation with MS analysis:

  • Use biotin-conjugated TNFAIP8L2 antibody to immunoprecipitate protein complexes

  • Analyze by mass spectrometry to identify binding partners

  • Validate key interactions by reverse co-IP and functional assays

  • Compare interactomes between resting and activated cells

• Yeast two-hybrid screening:

  • Use TNFAIP8L2 as bait to screen for interacting proteins

  • Validate positive hits in mammalian cells using co-IP

  • Map interaction domains through truncation or mutation studies

  • Current research has established interactions with RAC1 and competition with MTOR

• Structure-function analysis:

  • Generate domain-specific mutants (beyond the K15,16Q mutant)

  • Determine which domains are critical for different protein interactions

  • Correlate molecular interactions with functional outcomes in cell-based assays

  • Use findings to develop targeted intervention strategies

How can TNFAIP8L2 be effectively targeted in therapeutic development?

For therapeutic targeting approaches:

• Small molecule inhibitor development:

  • Focus on the RAC1-binding interface of TNFAIP8L2

  • Screen compound libraries for molecules that disrupt or enhance this interaction

  • Validate hits using interaction assays and functional readouts

  • Test promising compounds in inflammatory disease models

• Peptide-based strategies:

  • Design peptides that mimic critical binding domains

  • These peptides could compete with endogenous interactions

  • Modify for stability and cell penetration

  • Evaluate effects on RAC1-MTORC1 signaling and inflammatory responses

• Gene expression modulation:

  • Develop methods to normalize TNFAIP8L2 expression in disease states

  • For conditions with excessive inflammation, upregulate TNFAIP8L2

  • For immune suppression scenarios, consider TNFAIP8L2 inhibition

  • Validate approaches in appropriate disease models

• Combination therapy design:

  • Test TNFAIP8L2-targeting strategies alongside established treatments

  • For inflammatory conditions, combine with existing anti-inflammatory drugs

  • For metabolic disorders, pair with metabolic modulators

  • The dual immunological and metabolic roles of TNFAIP8L2 make it a promising target for complex diseases