TNFAIP8L2 Antibody, FITC conjugated

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

TNFAIP8L2 (Tumor Necrosis Factor Alpha-Induced Protein 8-Like 2), also known as TIPE2, is a 184 amino acid protein that functions as a critical negative regulator of both innate and adaptive immunity . It plays an essential role in maintaining immune homeostasis by regulating Toll-like receptor and T-cell receptor function . TIPE2 is preferentially expressed in lymphoid tissues including spleen, thymus, small intestine, and lymph nodes, with lower expression in colon, lung, and skin .

Research significance:

• Acts as a negative regulator that prevents excessive inflammatory responses

• Implicated in autoimmune disorders when downregulated

• Has emerging roles in tumor progression through RAS signaling pathway interaction

• Involved in cerebral ischemia inflammatory response

• Associated with diseases including skin squamous cell carcinoma

The protein's role in immune regulation makes TIPE2-targeting antibodies valuable tools for studying inflammation, autophagy, and immune response mechanisms.

What applications are FITC-conjugated TNFAIP8L2 antibodies validated for?

FITC-conjugated TNFAIP8L2 antibodies have been validated for multiple applications across various experimental systems:

For optimal results in immunofluorescence applications, researchers should begin with the recommended dilution range and optimize based on their specific experimental conditions, cell lines, and detection systems .

How should FITC-conjugated TNFAIP8L2 antibodies be stored to maintain reactivity?

Proper storage is crucial for maintaining antibody performance across multiple experiments:

Recommended storage conditions:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Some antibody preparations contain 50% glycerol to prevent freezing damage

• Protect from light exposure to prevent photobleaching of the FITC fluorophore

• Valid for approximately 12 months when stored properly

Practical methodology:

• Upon receipt, immediately aliquot the antibody into smaller volumes based on typical experimental usage (20-50 μl per aliquot)

• Store aliquots in opaque containers or wrapped in aluminum foil

• Thaw only the required amount for each experiment

• When working with the antibody, keep it on ice and protected from direct light

• Return to -20°C promptly after use

This storage protocol helps maintain the structural integrity of both the antibody and the FITC conjugate, ensuring consistent signal intensity across experiments .

How should researchers design proper controls when using FITC-conjugated TNFAIP8L2 antibodies?

Robust experimental design requires appropriate controls to validate specificity and reduce false positives:

Essential controls for immunofluorescence/flow cytometry:

For western blotting validation, additional controls using siRNA knockdown or CRISPR knockout of TNFAIP8L2 can further confirm antibody specificity .

What is the optimal sample preparation protocol for detecting TNFAIP8L2 in different cellular compartments?

TNFAIP8L2/TIPE2 localizes to multiple cellular compartments including cytoplasm and lysosomal membranes , requiring specific fixation and permeabilization protocols:

For immunofluorescence microscopy (cultured cells):

• Grow cells on sterile coverslips to 70-80% confluency

• Wash cells twice with PBS (pH 7.4)

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Wash 3x with PBS

• Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Block with 5% normal serum (from same species as secondary antibody) for 1 hour

• Incubate with FITC-conjugated TNFAIP8L2 antibody at 1:50-1:200 dilution overnight at 4°C

• Wash 3x with PBS

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

For tissue sections:

• For paraffin sections, antigen retrieval with TE buffer (pH 9.0) is recommended

• Alternatively, citrate buffer (pH 6.0) can be used

For lysosomal localization studies:

• Co-stain with lysosomal markers such as LAMP1 to visualize colocalization of TNFAIP8L2 with lysosomes

• Based on research findings, TNFAIP8L2 can localize to LAMP1-positive structures and affect lysosomal RAC1 levels

What are the optimal imaging parameters for FITC-conjugated TNFAIP8L2 in fluorescence microscopy?

Optimizing imaging parameters ensures high-quality data acquisition while minimizing photobleaching:

FITC fluorophore characteristics:

• Excitation maximum: ~495 nm

• Emission maximum: ~520 nm

• Filter set: Standard FITC/GFP filter set (typically 480/30 ex, 535/40 em)

Imaging recommendations:

• Exposure time: Start with short exposures (200-500ms) and adjust based on signal intensity

• Laser/lamp power: Use minimum power necessary to visualize signal (typically 10-30% of maximum)

• Gain settings: Begin with moderate gain settings and increase if necessary (trade-off with noise)

• Pixel dwell time (confocal): 1-2 μs for initial scans, increase for final images if needed

• Z-stack parameters: 0.3-0.5 μm step size for high-resolution 3D reconstruction

• Dynamic range: Capture using 12-16 bit depth for maximum information retention

• Sequential scanning: When performing multi-color imaging, use sequential scanning to prevent bleed-through

Advanced optimization:

• Employ deconvolution algorithms for improved signal-to-noise ratio

• For co-localization studies with lysosomal markers, consider super-resolution techniques such as STED or SIM microscopy to resolve structures below the diffraction limit

How can TNFAIP8L2 antibodies be used to investigate MTOR-dependent autophagy regulation?

Research has revealed that TNFAIP8L2 plays a critical role in autophagy regulation through the RAC1-MTOR axis, offering several experimental approaches:

Experimental design for studying TNFAIP8L2-autophagy regulation:

• Autophagic flux assessment:

  • Treat cells with TNFAIP8L2 overexpression/knockdown vectors

  • Monitor LC3B-II and SQSTM1 levels by western blotting

  • Use bafilomycin A1 (autophagosome-lysosome fusion inhibitor) to determine which step of autophagy is affected

• MTOR activity measurement:

  • Evaluate phosphorylation status of MTOR and its downstream targets (p-RPS6) by western blotting

  • Use rapamycin as a control for MTOR inhibition

• Visualization of autolysosome reformation:

  • Track LAMP1+LC3B+ puncta under starvation conditions

  • Monitor lysosome regeneration using LAMP+ vesicle quantification

  • Compare wild-type and TNFAIP8L2-overexpressing cells during prolonged starvation (8-10h)

• RAC1-MTOR-TNFAIP8L2 interaction studies:

  • Perform co-immunoprecipitation to assess RAC1-MTOR binding in presence/absence of TNFAIP8L2

  • Use immunofluorescence to visualize colocalization of RAC1 and MTOR on lysosomal membranes

Research has shown that TNFAIP8L2 competes with MTOR for binding to GTP-bound RAC1, thereby inhibiting MTORC1 activity during prolonged starvation conditions .

What methodological approaches can resolve contradictory data regarding TNFAIP8L2 localization patterns?

Researchers may encounter apparently contradictory data regarding TNFAIP8L2 subcellular localization due to various factors including cell type differences, experimental conditions, and technical limitations:

Resolution approaches:

• Multi-technique validation:

  • Combine immunofluorescence with subcellular fractionation and western blotting

  • Use both N and C-terminal targeting antibodies to account for potential cleavage products

  • Employ epitope-tagged constructs (GFP/FLAG-TNFAIP8L2) for verification

• Advanced imaging techniques:

  • Super-resolution microscopy (STED, STORM, SIM) to precisely define localization

  • Live-cell imaging with photoactivatable fluorescent protein fusions to track dynamic localization

  • Correlative light and electron microscopy for ultrastructural localization

• Context-dependent localization studies:

  • Examine localization under different cellular states:

    • Normal growth conditions

    • Starvation (amino acid/serum deprivation)

    • Inflammatory stimulation (LPS treatment)

    • Cell cycle phases

• Functional domain mapping:

  • Create deletion/mutation constructs to identify localization signal sequences

  • Particularly focus on the RAC1-binding domain (K15,16 region) which affects protein-protein interactions

Research indicates that TNFAIP8L2 can translocate between cytoplasmic and lysosomal compartments, necessitating careful experimental design to capture its dynamic localization patterns .

How can TNFAIP8L2 antibodies be applied in studies of immunometabolism and inflammation?

TNFAIP8L2/TIPE2 has emerged as a key regulator at the intersection of inflammation and metabolism, offering several research applications:

Experimental approaches:

• In vitro macrophage inflammation models:

  • Isolate bone marrow-derived macrophages (BMDMs) from wild-type and Tnfaip8l2-knockout mice

  • Stimulate with LPS to trigger inflammatory response

  • Measure pro-inflammatory cytokine production (IL6, TNF) by ELISA

  • Assess TNFAIP8L2 expression changes using the antibody

• Metabolic reprogramming analysis:

  • Measure mitochondrial respiration rate using Seahorse metabolic analyzer

  • Compare oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in wild-type versus Tnfaip8l2-deficient macrophages

  • Evaluate lipid biosynthesis pathway activation

• In vivo inflammation models:

  • LPS-induced endotoxemia model in wild-type and Tnfaip8l2-knockout mice

  • Monitor body temperature and clinical scores

  • Assess tissue inflammation via histology (H&E staining)

  • Measure circulating pro-inflammatory cytokines

  • Evaluate MTOR activity in tissues using p-RPS6 immunohistochemistry

• Gene expression profiling integration:

  • Perform RNA-seq on macrophages with varied TNFAIP8L2 expression

  • Analyze enrichment for inflammatory and metabolic pathways

  • Focus on leukocyte activation and lipid biosynthesis gene sets

Research has demonstrated that Tnfaip8l2 deficiency exacerbates inflammatory responses by upregulating MTOR activity, and TNFAIP8L2 serves as a "brake" for immunometabolism that must be released for effective inflammatory responses .

How can researchers troubleshoot weak or non-specific FITC-TNFAIP8L2 antibody signals in immunofluorescence?

Several factors can contribute to suboptimal staining results with FITC-conjugated TNFAIP8L2 antibodies:

Common issues and solutions:

Advanced troubleshooting:

• Perform titration experiments to determine optimal antibody concentration for specific cell types

• Consider alternative detection methods (e.g., indirect immunofluorescence with amplification)

• For co-localization studies, address potential fluorophore interactions and spectral overlap

What quality control metrics should be established for FITC-conjugated TNFAIP8L2 antibody validation?

Implementing comprehensive quality control is essential for reliable TNFAIP8L2 antibody applications:

Validation metrics:

• Specificity assessment:

  • Western blot showing single band at expected molecular weight (18-20 kDa)

  • Absence of signal in TNFAIP8L2 knockout/knockdown samples

  • Signal reduction in peptide competition assays

  • Consistent staining pattern across multiple antibody clones targeting different epitopes

• Sensitivity measurements:

  • Limit of detection determination using serial dilutions of recombinant protein

  • Signal-to-noise ratio quantification in immunofluorescence images

  • Dynamic range assessment across expression levels

• Reproducibility standards:

  • Intra-assay variability (<10% CV preferred)

  • Inter-assay variability (<15% CV preferred)

  • Lot-to-lot consistency validation

• Application-specific validation:

  • For flow cytometry: Staining index calculation, resolution of positive/negative populations

  • For microscopy: Colocalization coefficients with known markers (e.g., LAMP1 for lysosomal localization)

  • For quantitative applications: Standard curve linearity, dynamic range assessment

Documentation best practices:

• Maintain detailed records of validation experiments including all controls

• Include positive control samples in each experimental run

• Document lot numbers and validation data for antibody batches

How can live-cell imaging approaches be optimized for studying TNFAIP8L2 dynamics during autophagy?

Live-cell imaging offers unique insights into the dynamic role of TNFAIP8L2 in autophagy regulation, particularly during autolysosome reformation:

Methodological approach:

• Expression construct design:

  • Create fluorescent protein fusion constructs (e.g., TNFAIP8L2-mEGFP or mCherry-TNFAIP8L2)

  • Validate fusion protein functionality through rescue experiments in TNFAIP8L2-knockout cells

  • Consider using CRISPR knock-in approaches for endogenous tagging to maintain physiological expression levels

• Multi-color imaging system:

  • Combine with lysosomal markers (LAMP1-mRFP) and autophagosomal markers (GFP-LC3 or mCherry-GFP-LC3 tandem reporter)

  • Use spectrally compatible fluorophores to minimize bleed-through

  • Design time-lapse experiments capturing:

    • Starvation-induced autophagy induction

    • Autolysosome formation

    • Autolysosome reformation during prolonged starvation

• Acquisition parameters optimization:

  • Minimize phototoxicity: Reduce light intensity, increase camera sensitivity

  • Balance temporal resolution with photobleaching (typically 1-5 minute intervals)

  • Employ spinning disk confocal for improved signal-to-noise ratio with less phototoxicity

  • Use environmental chamber to maintain 37°C, 5% CO₂, and humidity

• Advanced analytical approaches:

  • Particle tracking algorithms to follow vesicle dynamics

  • Intensity-based colocalization analysis over time

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

  • FRET-based assays to examine TNFAIP8L2-RAC1 interactions in real-time

Research has shown that TNFAIP8L2 impairs autolysosome reformation during prolonged starvation, suggesting its dynamic involvement in different phases of the autophagy process .

How can TNFAIP8L2 antibodies be integrated into multi-parameter flow cytometry panels for immune phenotyping?

TNFAIP8L2's role as an immune regulator makes it valuable in comprehensive immune phenotyping panels:

Panel design considerations:

• Cell surface marker combinations:

  • Combine FITC-TNFAIP8L2 with markers for myeloid populations (CD11b, CD14, CD16)

  • Include T-cell markers (CD3, CD4, CD8) and activation markers (CD69, CD25)

  • Add lineage markers for dendritic cells, NK cells, and B cells as needed

• Intracellular staining protocol optimization:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% saponin buffer or commercial permeabilization reagents

  • Block with 2% normal serum

  • Stain with FITC-TNFAIP8L2 antibody (0.40 μg per 10^6 cells)

  • Include cytokine staining (IL-6, TNF, IL-10) for functional assessment

• Spectral considerations:

  • FITC spectrum (excitation ~495nm, emission ~520nm) may overlap with PE

  • Consider alternative conjugates (AF488) if panel design requires PE

  • Use compensation controls for each fluorochrome

  • When possible, place TNFAIP8L2 on a separate laser line from potentially overlapping fluorophores

• Analysis strategies:

  • Gate on specific immune populations first, then assess TNFAIP8L2 expression

  • Use biaxial plots, histogram overlays, and dimensionality reduction (tSNE, UMAP)

  • Correlate TNFAIP8L2 expression with activation markers and cytokine production

  • Compare expression across disease states or treatment conditions

Example applications:

• Monitor TNFAIP8L2 expression changes in inflammatory diseases

• Assess correlation between TNFAIP8L2 levels and MTOR pathway activation markers

• Evaluate TNFAIP8L2 expression in tumor-infiltrating immune cells

What approaches can integrate TNFAIP8L2 antibody-based studies with omics data for systems biology analysis?

Integrating antibody-based protein detection with multi-omics datasets enables systems-level understanding of TNFAIP8L2 function:

Integration methodologies:

• Proteogenomic correlation:

  • Measure TNFAIP8L2 protein levels via IF/WB in multiple cell states

  • Perform parallel RNA-seq to correlate transcript and protein expression

  • Identify post-transcriptional regulation mechanisms

  • Use genome-wide gene expression profiling to identify enriched pathways

• Protein interactome mapping:

  • Use TNFAIP8L2 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Validate key interactions (e.g., RAC1, MTOR) via co-IP and IF colocalization

  • Build interaction networks incorporating known binding partners

  • Overlay with phosphoproteomic data to identify signaling nodes

• Functional genomics integration:

  • Combine CRISPR-Cas9 genetic screens with immunophenotyping

  • Identify genetic modifiers of TNFAIP8L2 function in autophagy and immune regulation

  • Use antibodies to validate screen hits via protein expression/localization changes

• Computational approaches:

  • Network analysis of TNFAIP8L2-centered protein-protein interactions

  • Pathway enrichment analysis of differentially expressed genes in TNFAIP8L2-deficient cells

  • Machine learning classification of cellular phenotypes based on TNFAIP8L2 expression patterns

Research applications:

• Map TNFAIP8L2's role in leukocyte activation and lipid biosynthesis pathways

• Identify novel regulatory mechanisms connecting metabolism and inflammation

• Develop predictive models for inflammatory disease progression based on TNFAIP8L2 network status

How can TNFAIP8L2 antibodies contribute to preclinical evaluation of immunotherapeutic approaches?

TNFAIP8L2's role as a negative regulator of immunity positions it as a potential therapeutic target and biomarker:

Preclinical research applications:

• Biomarker development:

  • Quantify TNFAIP8L2 expression in patient-derived samples via flow cytometry

  • Correlate expression levels with disease severity or treatment response

  • Develop standardized immunoassays for clinical sample analysis

  • Evaluate TNFAIP8L2 as a predictive biomarker for immunotherapy response

• Therapeutic target validation:

  • Use antibodies to monitor target engagement in drug development

  • Assess TNFAIP8L2 expression changes following treatment with immune modulators

  • Evaluate effects of TNFAIP8L2 modulation on immune activation and inflammation

  • Investigate TNFAIP8L2 as a drug target for inflammatory diseases

• In vivo efficacy studies:

  • Monitor TNFAIP8L2 expression in immune cells from treated animals

  • Correlate with inflammatory markers, disease scores, and survival outcomes

  • Evaluate tissue-specific changes in protein expression

  • Assess MTOR pathway activation status in response to TNFAIP8L2 modulation

• Mechanism of action studies:

  • Use multiplexed IF to evaluate TNFAIP8L2 alongside key signaling nodes

  • Investigate autophagy modulation as a mechanism in anti-tumor immunity

  • Assess metabolic reprogramming in immune cells following TNFAIP8L2 manipulation

Research has demonstrated that TNFAIP8L2 deficiency exacerbates inflammatory responses and lung injury in endotoxemia models by upregulating MTOR activity, suggesting its potential as a therapeutic target for inflammatory conditions .

What are the critical differences between various FITC-conjugated TNFAIP8L2 antibody preparations?

Researchers should be aware of important variations between commercially available FITC-conjugated TNFAIP8L2 antibodies:

Key parameters to compare:

Critical considerations:

• Antibodies targeting different epitopes may yield varying results in detecting TNFAIP8L2 complexes with binding partners like RAC1

• The K15,16 region is critical for RAC1 binding; antibodies targeting this region may interfere with protein-protein interactions

• Cross-reactivity with other TNFAIP8 family members should be assessed, particularly in systems expressing multiple family members

How can multi-color immunofluorescence be optimized for studying TNFAIP8L2 interactions with the autophagy machinery?

TNFAIP8L2's complex role in autophagy regulation involves interactions with multiple proteins that can be visualized through optimized multi-color immunofluorescence:

Protocol optimization:

• Antibody panel design:

  • FITC-TNFAIP8L2 antibody (primary or directly conjugated)

  • Markers for autophagosomes: LC3B (different fluorophore)

  • Lysosomal markers: LAMP1

  • MTOR pathway components: mTOR, phospho-S6

  • RAC1 (binding partner of TNFAIP8L2)

• Sequential staining approach:

  • Begin with the lowest concentration antibody

  • Use sequential rather than cocktail staining to minimize cross-reactivity

  • Include blocking steps between antibody applications

  • Consider tyramide signal amplification for low abundance targets

• Advanced fixation techniques:

  • For membrane protein preservation: Use mild fixation (1-2% PFA) or glyoxal-based fixatives

  • For detecting transient interactions: Consider proximity ligation assay (PLA)

  • For ultrastructural localization: Correlative light and electron microscopy

• Imaging considerations:

  • Use confocal microscopy with appropriate channel settings

  • Consider spectral unmixing for closely overlapping fluorophores

  • Apply deconvolution algorithms to improve resolution

  • For co-localization analysis, acquire images at Nyquist sampling rate

Research applications:

• Visualize RAC1-MTOR-TNFAIP8L2 interactions on lysosomal membranes

• Track autolysosome reformation during prolonged starvation

• Assess TNFAIP8L2 competition with MTOR for RAC1 binding

What methodological advances can improve detection sensitivity for low abundance TNFAIP8L2 in tissue samples?

Detecting low-abundance TNFAIP8L2 in tissues with variable expression levels requires specialized approaches:

Advanced detection strategies:

• Signal amplification methods:

  • Tyramide signal amplification (TSA): Enhances FITC signal up to 100-fold

  • Implementation protocol:

    • Use HRP-conjugated secondary antibody

    • Apply FITC-tyramide substrate

    • HRP converts tyramide to reactive intermediate that covalently binds nearby proteins

    • Results in localized signal amplification

• Optimized antigen retrieval:

  • Pressure cooker-based retrieval in TE buffer (pH 9.0)

  • Extended retrieval times (20-40 minutes)

  • Use of commercial epitope retrieval solutions with proprietary buffers

  • Alternative retrieval methods for difficult tissues:

    • Protease-induced epitope retrieval

    • Heat-induced epitope retrieval in different buffers

• Detection system improvements:

  • Quantum dot-conjugated systems for enhanced photostability

  • Use of high-sensitivity sCMOS or EMCCDs for imaging

  • Computational approaches:

    • Deconvolution algorithms

    • Machine learning-based signal enhancement

    • Background subtraction techniques

• Pre-analytical considerations:

  • Minimize time from tissue collection to fixation

  • Optimize fixation duration (12-24h recommended)

  • Use consistent sectioning thickness (4-6μm optimal)

  • Apply tissue-specific blocking reagents to reduce background

Research applications include detecting TNFAIP8L2 in tissues with naturally low expression (colon, lung, skin) and tracking expression changes in disease states such as inflammatory conditions or cancer .