IL13RA1 Antibody, HRP conjugated

What is IL13RA1 and what role does it play in immune signaling?

IL13RA1 (Interleukin 13 Receptor Subunit Alpha 1) is a type 1 membrane protein belonging to the hematopoietin receptor family. It functions as a subunit of the interleukin 13 receptor, forming a receptor complex with the IL-4 receptor alpha (IL4RA) subunit. This complex is crucial for both IL-13 and IL-4 signaling pathways . IL13RA1 serves as the primary IL-13-binding component of the IL-13 receptor. At the molecular level, IL13RA1 has been shown to bind tyrosine kinase TYK2, mediating signaling processes that lead to the activation of JAK1, STAT3, and STAT6 when induced by either IL-13 or IL-4 .

The human IL13RA1 cDNA encodes a 427 amino acid precursor protein with a 21 amino acid signal peptide, 324 amino acid extracellular domain, 23 amino acid transmembrane region, and a 59 amino acid cytoplasmic tail . This receptor is particularly important in allergic inflammation and immune response to parasite infections. IL13RA1 combines with IL-4 receptor alpha to form a high-affinity receptor complex capable of transducing IL-13-dependent proliferative signals .

How do HRP-conjugated antibodies function in detection systems?

HRP (Horseradish Peroxidase)-conjugated antibodies function as detection tools in various immunoassays by combining the specific binding properties of antibodies with the enzymatic activity of HRP. When the antibody portion binds to its target antigen (in this case IL13RA1), the conjugated HRP enzyme can catalyze a reaction with an appropriate substrate, producing a detectable signal.

In Western blot applications, HRP-conjugated anti-IL13RA1 antibodies can be used to directly detect the protein of interest after separation by gel electrophoresis and transfer to a membrane . Alternatively, an unconjugated primary anti-IL13RA1 antibody can be used followed by an HRP-conjugated secondary antibody, as demonstrated in the detection protocol where a PVDF membrane was probed with Mouse Anti-Human IL-13 Ra1 Monoclonal Antibody followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody .

The enzymatic reaction catalyzed by HRP typically involves oxidation of substrates like luminol (for chemiluminescence detection) or DAB (3,3'-diaminobenzidine, for colorimetric detection). The resulting signal can be captured via X-ray film, digital imaging systems, or visual observation depending on the substrate used.

What are the structural characteristics of IL13RA1 that affect antibody selection?

The structural features of IL13RA1 are critical considerations when selecting appropriate antibodies:

• Extracellular domain: IL13RA1 has a 324 amino acid extracellular domain (from Ala27 to Thr343 in humans) that represents the primary target for most commercial antibodies . Antibodies targeting this region are particularly useful for applications like flow cytometry where cell surface detection is required.

• Homology considerations: Human and mouse IL13RA1 share 76% amino acid sequence identity . This relatively high conservation means some antibodies may cross-react between species, but species-specific validation is essential for research applications crossing these boundaries.

• Relationship to IL13RA2: The extracellular domain of IL13RA1 is closely related to that of IL13RA2 . This structural similarity creates potential for cross-reactivity, so antibodies should be validated for specificity against both receptors.

• Phosphorylation sites: Functional IL13RA1 contains important phosphorylation sites, such as Tyr405, which are crucial for signaling . When studying receptor activation or signaling, phospho-specific antibodies targeting these sites are available.

When selecting HRP-conjugated IL13RA1 antibodies, researchers should consider which epitopes are accessible in their experimental system and whether post-translational modifications might affect antibody binding.

What are the optimal conditions for using IL13RA1 HRP-conjugated antibodies in Western blotting?

For optimal results with IL13RA1 HRP-conjugated antibodies in Western blotting, consider the following methodological approach:

• Sample preparation: For cell lysates, use RIPA buffer supplemented with protease inhibitors. When working with tissues, homogenization in a suitable lysis buffer followed by clarification through centrifugation is recommended.

• Protein loading: Load 20-40 μg of total protein per lane, depending on IL13RA1 expression levels in your sample. Overloading can lead to background issues.

• Membrane selection: PVDF membranes have been successfully used for IL13RA1 detection as demonstrated in published protocols . PVDF typically provides better protein retention compared to nitrocellulose for this application.

• Blocking conditions: Block with 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature.

• Antibody concentration: For direct HRP-conjugated antibodies, a concentration of 1-2 μg/mL is typically effective, though this should be optimized for each specific antibody .

• Incubation parameters: Incubate with primary antibody overnight at 4°C or for 2 hours at room temperature with gentle agitation.

• Washing steps: Wash 3-5 times with TBST, 5 minutes per wash, to reduce background.

• Detection system: Use enhanced chemiluminescence (ECL) substrates appropriate for the expected signal intensity. Low abundance proteins may require more sensitive substrates.

• Exposure optimization: Start with short exposures (30 seconds) and increase as needed to obtain optimal signal-to-noise ratio.

• Controls: Include positive controls (such as Myc-tagged IL13RA1-transfected 293T cells) and negative controls to validate specificity .

How should researchers validate IL13RA1 antibody specificity before experimental use?

Proper validation of IL13RA1 antibody specificity is essential for reliable experimental outcomes and should include:

• Positive control samples: Use cells or tissues known to express IL13RA1, such as human peripheral blood granulocytes, which have been successfully used to detect IL13RA1 by flow cytometry .

• Knockdown/knockout verification: Whenever possible, compare antibody reactivity in wild-type samples versus those with IL13RA1 knockdown or knockout to confirm specificity.

• Recombinant protein testing: Test antibody against purified recombinant IL13RA1 protein to confirm binding to the intended target.

• Cross-reactivity assessment: Verify lack of binding to related proteins, particularly IL13RA2, which shares structural similarities with IL13RA1 .

• Isotype controls: Use appropriate isotype control antibodies to identify non-specific binding, as demonstrated in flow cytometry protocols where isotype control antibodies (e.g., Catalog # IC0041A, IC0041P) were used alongside test antibodies .

• Peptide competition: Perform peptide competition assays where pre-incubation with the immunizing peptide should abolish specific staining.

• Multiple detection methods: Validate using complementary techniques (e.g., if using for Western blot, confirm with immunoprecipitation or immunofluorescence).

• Species cross-reactivity testing: If working across species, verify reactivity in each relevant species. For IL13RA1, predicted reactivity has been noted for rat (80%), pig (82%), and bovine (89%) in addition to human and mouse models .

• Lot-to-lot consistency: When receiving new antibody lots, perform comparative validation against previous lots to ensure consistent performance.

What flow cytometry protocols are recommended for IL13RA1 detection?

For flow cytometric detection of IL13RA1, the following optimized protocol is recommended based on published methods:

• Cell preparation: Isolate target cells (e.g., peripheral blood granulocytes) using standard isolation protocols. Ensure high viability (>90%) for reliable results.

• Cell count and washing: Adjust cell concentration to 1×10^6 cells per 100 μL in flow cytometry buffer (PBS containing 0.5% BSA and 2 mM EDTA). Wash cells twice with this buffer.

• Fc receptor blocking: Incubate cells with Fc receptor blocking solution for 10 minutes at room temperature to minimize non-specific binding.

• Surface staining: For IL13RA1 detection, use fluorophore-conjugated antibodies such as PE-conjugated (Catalog # FAB1462P) or APC-conjugated (Catalog # FAB1462A) anti-IL13RA1 antibodies at manufacturer-recommended concentrations (typically 5-10 μL per million cells) .

• Co-staining markers: Include appropriate lineage markers for identifying specific cell populations. For human granulocytes, Siglec-3/CD33 has been successfully used (PE-conjugated, Catalog # FAB1137P or APC-conjugated, Catalog # FAB1137A) .

• Incubation parameters: Stain cells for 30 minutes at 2-8°C in the dark.

• Washing after staining: Wash cells twice with flow cytometry buffer.

• Viability dye: Include an appropriate viability dye to exclude dead cells from analysis.

• Controls: Always include:

  • Unstained controls

  • Single-stained controls for compensation setup

  • FMO (Fluorescence Minus One) controls

  • Isotype controls (Catalog # IC0041A or IC0041P have been used for IL13RA1 antibodies)

• Data acquisition: Acquire at least 10,000 events within the population of interest.

• Analysis approach: Use bivariate dot plots to analyze co-expression patterns. Histogram overlays can be used to compare IL13RA1 expression between different populations or between samples and isotype controls.

This protocol has been verified for detecting IL13RA1 in human blood granulocytes and can be adapted for other cell types with appropriate modifications .

What are common sources of background when using IL13RA1 HRP-conjugated antibodies?

When using IL13RA1 HRP-conjugated antibodies, several factors can contribute to background issues:

• Insufficient blocking: Inadequate blocking of non-specific binding sites on membranes or in cell preparations is a primary cause of background. Optimize blocking conditions using different blockers (BSA, casein, commercial blockers) and concentrations.

• Cross-reactivity: Due to the structural similarity between IL13RA1 and IL13RA2, antibodies may exhibit cross-reactivity . Validate specificity using samples with known expression profiles of both receptors.

• Endogenous peroxidase activity: In tissue sections or certain cell types, endogenous peroxidase activity can cause false-positive signals. Include a peroxidase quenching step (e.g., 3% H₂O₂ treatment) before antibody application.

• Antibody concentration: Excessive antibody concentration increases non-specific binding. Titrate antibodies to determine optimal working dilutions for each application and sample type.

• Secondary antibody cross-reactivity: If using an indirect detection method, secondary antibodies may bind non-specifically to endogenous immunoglobulins in samples. Pre-adsorbed secondary antibodies specific to the host species of the primary antibody can minimize this issue.

• Inadequate washing: Insufficient washing between steps leads to reagent carryover and increased background. Implement more stringent washing steps (increased duration, volume, or number of washes).

• Sample-specific factors: Certain tissues or cell lines may contain proteins that non-specifically bind antibodies. Sample-specific optimization may be necessary.

• Substrate exposure time: Excessive substrate reaction time with HRP can lead to non-specific signal development. Optimize exposure times for each experimental setup.

• Antibody storage and handling: Improper storage or repeated freeze-thaw cycles can cause antibody degradation leading to non-specific binding. Follow manufacturer guidelines for storage conditions, which typically recommend 2-8°C and protection from light for conjugated antibodies .

How can researchers optimize signal-to-noise ratio when using IL13RA1 HRP-conjugated antibodies?

To optimize signal-to-noise ratio with IL13RA1 HRP-conjugated antibodies:

• Antibody titration: Perform a systematic titration series to determine the minimum antibody concentration that yields maximum specific signal with minimal background.

• Buffer optimization: Test different buffer compositions (varying salt concentrations, detergent types and concentrations, pH) to identify conditions that maximize specific binding while minimizing non-specific interactions.

• Blocking optimization: Compare different blocking agents (BSA, non-fat dry milk, commercial blockers) at various concentrations and incubation times to identify optimal conditions for your specific sample type.

• Enhanced washing protocols: Implement more stringent washing steps between incubations, potentially including higher detergent concentrations or additional wash steps.

• Sample preparation refinement: Optimize protein extraction and sample preparation methods to reduce interfering substances. For cell lysates, compare different lysis buffers and clarification methods.

• Signal amplification systems: For low-abundance targets, consider using signal amplification methods compatible with HRP detection, such as tyramide signal amplification (TSA).

• Substrate selection: Different HRP substrates offer varying levels of sensitivity and signal duration. For Western blotting, enhanced chemiluminescence (ECL) substrates with different sensitivity levels are available and should be selected based on target abundance.

• Incubation conditions: Optimize temperature and duration of antibody incubations. Longer incubations at 4°C often provide better signal-to-noise ratios than shorter incubations at room temperature.

• Membrane selection for Western blotting: Compare different membrane types (PVDF vs. nitrocellulose) and pore sizes to optimize protein retention and antibody accessibility.

• Sample dilution series: Run a dilution series of your sample to identify the optimal loading amount that provides sufficient signal without saturation or increased background.

• Reagent quality control: Ensure all reagents are fresh and properly stored. HRP-conjugated antibodies should be protected from light and stored at 2-8°C (not frozen) .

What are essential controls for experiments using IL13RA1 HRP-conjugated antibodies?

When conducting experiments with IL13RA1 HRP-conjugated antibodies, the following controls are essential for result validation:

Positive Controls:

• Known positive samples: Include cell lines or tissues with confirmed IL13RA1 expression. Human peripheral blood granulocytes have been validated for IL13RA1 expression . For Western blotting, Myc-tagged IL13RA1-transfected 293T cells serve as excellent positive controls .

• Recombinant IL13RA1 protein: Use purified recombinant IL13RA1 protein (particularly the extracellular domain, Ala27-Thr343) as a standard for antibody performance verification .

Negative Controls:

• Isotype controls: For flow cytometry, include appropriate isotype control antibodies matched to the primary antibody's isotype, host species, and conjugate (e.g., Catalog # IC0041A for APC-conjugated or IC0041P for PE-conjugated antibodies) .

• IL13RA1-negative samples: Include cell lines or samples known not to express IL13RA1 to confirm specificity.

• Knockdown/knockout samples: When available, use IL13RA1 knockdown or knockout samples as definitive negative controls.

Technical Controls:

• Secondary antibody only: For indirect detection systems, include controls with secondary antibody only (no primary) to assess non-specific binding.

• Blocking peptide competition: Pre-incubate antibody with the immunizing peptide before application to demonstrate binding specificity.

• Dilution series: Include a dilution series of your sample to demonstrate signal proportionality to target concentration.

• Substrate-only control: Include a lane or sample treated with substrate but no antibody to assess endogenous enzyme activity or substrate stability issues.

Process Controls:

• Loading controls: For Western blots, include housekeeping protein detection (β-actin, GAPDH) to normalize for loading variations.

• Internal reference standards: Include samples with known IL13RA1 expression levels across experiments to monitor inter-assay variability.

• Multi-parameter controls: For flow cytometry, include established markers like Siglec-3/CD33 for population identification and to validate staining protocol functionality .

• Cross-reactivity assessment: Test the antibody against related proteins, particularly IL13RA2, to confirm specificity .

How can IL13RA1 antibodies be used to investigate IL-13/IL-4 signaling pathway interactions?

IL13RA1 antibodies provide powerful tools for investigating the complex IL-13/IL-4 signaling pathways through several advanced approaches:

• Receptor complex formation analysis: IL13RA1 combines with IL-4 receptor alpha to form a high-affinity receptor complex capable of transducing both IL-13 and IL-4 signals . Use co-immunoprecipitation with IL13RA1 HRP-conjugated antibodies to pull down the receptor complex, followed by immunoblotting for IL4RA to quantify complex formation under different conditions.

• Signaling pathway activation studies: IL13RA1 mediates signaling that activates JAK1, STAT3, and STAT6 . Use IL13RA1 antibodies in combination with phospho-specific antibodies against these downstream molecules to track signaling cascade activation in response to ligand binding or pathway manipulation.

• Receptor phosphorylation detection: Utilize phospho-specific antibodies targeting key phosphorylation sites on IL13RA1, such as Tyr405, to monitor receptor activation status . This approach can help determine the kinetics and magnitude of receptor activation under various experimental conditions.

• Ligand competition assays: Both IL-13 and IL-4.can utilize the IL13RA1/IL4RA complex. Employ IL13RA1 antibodies in competitive binding assays to investigate how these cytokines compete for receptor binding, potentially using fluorescently-labeled cytokines and flow cytometry to quantify binding in the presence of blocking antibodies.

• Receptor trafficking studies: Combine IL13RA1 antibodies with subcellular fractionation or imaging techniques to track receptor internalization and recycling following ligand binding. This can provide insights into signal termination mechanisms.

• Functional blocking experiments: Use neutralizing IL13RA1 antibodies to selectively block receptor function and assess the differential effects on IL-13 versus IL-4 signaling, helping to delineate unique versus shared pathway components.

• Interaction with TYK2: IL13RA1 has been shown to bind tyrosine kinase TYK2 . Use IL13RA1 antibodies in proximity ligation assays or FRET-based approaches to visualize and quantify these interactions in intact cells.

• Heterodimer versus homodimer analysis: Investigate potential receptor clustering by using differently-labeled IL13RA1 antibodies in combination with super-resolution microscopy to determine receptor organization at the cell surface.

What methodological approaches can be used to study IL13RA1 in allergic inflammation models?

Studying IL13RA1 in allergic inflammation models requires specialized methodological approaches:

• Tissue-specific expression profiling: Use HRP-conjugated IL13RA1 antibodies for immunohistochemistry to map receptor expression across tissues relevant to allergic inflammation (lung, skin, gastrointestinal tract). Compare expression patterns between healthy and inflamed tissues to identify regulation patterns.

• Cell-specific expression analysis: Implement multi-parameter flow cytometry using fluorochrome-conjugated IL13RA1 antibodies combined with lineage markers to identify which immune cell populations express IL13RA1 during allergic responses. Human peripheral blood granulocytes provide a validated starting point for such analyses .

• Receptor occupancy assays: Develop assays using non-competing IL13RA1 antibodies to measure what fraction of receptors are occupied by endogenous ligand during inflammatory responses, providing insights into pathway activation levels.

• Temporal regulation studies: Deploy IL13RA1 antibodies in time-course experiments to track receptor expression and activation during different phases of allergic inflammation (sensitization, acute response, chronic inflammation, resolution).

• In vivo imaging approaches: Adapt IL13RA1 antibodies for in vivo imaging by conjugating to appropriate imaging agents (fluorescent dyes, PET tracers) to visualize receptor dynamics in live animal models of allergic inflammation.

• Correlation with clinical parameters: In human studies, combine IL13RA1 expression analysis using validated antibodies with measurements of clinical parameters (lung function, symptom scores) to establish relationships between receptor expression and disease severity.

• Genetic model validation: Use IL13RA1 antibodies to confirm altered expression in genetic models (receptor knockouts, conditional deletions) and correlate with functional outcomes in allergic challenge models.

• Therapeutic intervention assessment: Employ IL13RA1 antibodies to monitor receptor expression and occupancy before and after therapeutic interventions targeting the IL-13/IL-4 pathway, helping to establish pharmacodynamic biomarkers.

• Single-cell analysis: Integrate IL13RA1 antibody-based detection with single-cell technologies (mass cytometry, single-cell RNA-seq) to resolve heterogeneity in receptor expression and correlate with cellular phenotypes at high resolution.

How does IL13RA1 differ functionally from IL13RA2, and what methodological approaches can distinguish them?

IL13RA1 and IL13RA2 have distinct functional properties despite structural similarities, requiring specific methodological approaches to distinguish them:

Functional Differences:

• Signaling capacity: IL13RA1 combines with IL4RA to form a functional signaling complex that activates JAK1, STAT3, and STAT6 . In contrast, IL13RA2 primarily functions as a decoy receptor, binding IL-13 with high affinity but generally not activating canonical signaling pathways .

• Ligand affinity: IL13RA2 binds IL-13 with higher affinity than IL13RA1 does . IL13RA1 requires partnership with IL4RA to form a high-affinity complex for IL-13.

• Cytoplasmic domain: IL13RA1 has a 59 amino acid cytoplasmic domain capable of interacting with signaling molecules like TYK2 . IL13RA2 has a much shorter cytoplasmic tail with limited signaling capacity.

• Receptor partners: IL13RA1 forms functional heterodimers with IL4RA , while IL13RA2 typically functions independently.

• Internalization behavior: IL13RA2 mediates internalization and depletion of extracellular IL-13, serving as a negative regulator .

Methodological Approaches to Distinguish Them:

• Epitope-specific antibodies: Develop and validate antibodies targeting non-conserved epitopes unique to each receptor. Despite the relatedness of their extracellular domains , there are distinct regions that can be exploited for specific antibody development.

• Phospho-specific detection: Use phospho-specific antibodies targeting sites unique to IL13RA1 (such as pY405) to distinguish active IL13RA1 signaling from IL13RA2.

• Co-immunoprecipitation strategies: Perform co-immunoprecipitation with IL4RA-specific antibodies, which should pull down IL13RA1 but not IL13RA2 due to their different partnering behaviors.

• Functional readouts: Implement reporter assays measuring STAT6 activation as a functional readout of IL13RA1 activity, which would not be triggered by IL13RA2.

• Kinetic binding studies: Use surface plasmon resonance or similar techniques with purified receptors to distinguish the different binding kinetics of IL-13 to each receptor.

• Cellular localization analysis: Perform subcellular fractionation followed by immunoblotting or confocal microscopy with specific antibodies to map different distribution patterns between the receptors.

• Receptor depletion strategies: Use siRNA or CRISPR approaches to selectively deplete each receptor and confirm specificity of antibody detection methods.

• Ligand interaction analysis: Develop competition assays where labeled IL-13 binding is assessed in the presence of blocking antibodies specific for each receptor subtype.

• In situ hybridization: Use RNA probes targeting unique sequence regions to distinguish expression patterns at the mRNA level before confirming with protein detection.

• Mass spectrometry: Implement immunoprecipitation followed by mass spectrometry to identify unique peptides that can distinguish between the two receptors.

What experimental design considerations are important when using IL13RA1 antibodies in multiplex analysis systems?

When incorporating IL13RA1 antibodies into multiplex analysis systems, several critical experimental design considerations must be addressed:

• Antibody compatibility assessment: Before multiplexing, validate that IL13RA1 antibodies do not cross-react with other targets in your panel. This is particularly important for closely related family members like IL13RA2 .

• Fluorophore selection for flow cytometry: When selecting fluorophore-conjugated IL13RA1 antibodies (like APC-conjugated or PE-conjugated ), consider the brightness hierarchy and spectral overlap with other panel components. Place antibodies detecting low-abundance targets on brighter fluorophores.

• Panel design optimization:

  • Balance panel by spreading bright and dim fluorophores across detection channels

  • Account for antigen density (IL13RA1 expression levels vary by cell type)

  • Consider potential fluorescence spillover between channels

  • Include a dead cell exclusion marker compatible with other fluorophores

• Compensation controls: For flow cytometry applications, prepare single-stained controls for each fluorophore using the same antibody concentrations as in the full panel. Anti-IL13RA1 antibodies conjugated to APC or PE have been successfully used in flow cytometry panels alongside other markers like Siglec-3/CD33 .

• Antibody titration in multiplex context: Re-titrate IL13RA1 antibodies in the presence of the complete antibody panel, as optimal concentrations may differ from those determined in single-staining experiments.

• Order of antibody application: In sequential staining protocols, determine optimal staining sequence to prevent epitope blocking or steric hindrance between antibodies.

• Validation of multiplex results: Confirm key findings from multiplex experiments using conventional single-target methods to verify that multiplexing does not affect detection accuracy.

• Sample preprocessing considerations: For complex samples (tissue homogenates, clinical specimens), optimize sample preparation methods to ensure consistent accessibility of all target epitopes.

• Detection system compatibility: When using HRP-conjugated IL13RA1 antibodies in multiplex immunoassays, ensure compatibility with other detection enzymes in the system to prevent cross-reactivity or signal interference.

• Data normalization strategy: Develop appropriate normalization approaches that account for differences in detection sensitivity across targets in the multiplex panel.

• Reference standards: Include multiparameter reference standards that express known levels of IL13RA1 alongside other panel targets to enable accurate quantification and inter-assay comparability.

• Batch effects mitigation: Implement strategies to identify and correct for batch effects in large-scale multiplex studies, particularly important for clinical or longitudinal research using IL13RA1 as a biomarker.

How can IL13RA1 antibodies contribute to understanding allergic inflammation mechanisms?

IL13RA1 antibodies are invaluable tools for unraveling allergic inflammation mechanisms through several methodological approaches:

• Cell-specific expression profiling: IL13RA1 is expressed on various cell types involved in allergic responses. Using flow cytometry with fluorophore-conjugated IL13RA1 antibodies (such as PE-conjugated or APC-conjugated) allows precise quantification of receptor expression across immune cell populations, particularly in granulocytes which have been well-characterized for IL13RA1 expression .

• Signaling pathway dissection: IL13RA1 is a critical component of the IL-13 signaling pathway that drives allergic inflammation. IL13RA1 forms a complex with IL4RA to transduce IL-13 signals , activating JAK1, STAT3, and STAT6 . Using antibodies against both the receptor and its phosphorylated forms enables tracking of signal transduction dynamics in allergic models.

• Functional blocking studies: Neutralizing antibodies against IL13RA1 can be used to selectively block receptor function in experimental systems, helping differentiate between IL-13-dependent and independent processes in allergic inflammation.

• Therapeutic target validation: IL-13 is a key mediator of allergic inflammation. Antibodies detecting IL13RA1 can be used to validate receptor expression in target tissues before and after experimental therapeutic interventions.

• Tissue microenvironment analysis: Immunohistochemistry using HRP-conjugated IL13RA1 antibodies enables visualization of receptor distribution within allergic tissues, providing spatial context for understanding local signaling dynamics.

• Receptor-ligand interaction studies: IL13RA1 antibodies can be used in competitive binding assays to investigate how IL-13 engages its receptor under different inflammatory conditions, potentially identifying factors that modulate this interaction during allergic responses.

• Cross-talk with other pathways: Co-immunoprecipitation using IL13RA1 antibodies followed by proteomic analysis can identify novel interaction partners that may represent unexplored components of allergic signaling networks.

• Biomarker development: Quantitative assays using IL13RA1 antibodies can assess receptor levels in patient samples, potentially yielding biomarkers that correlate with allergic disease severity or therapeutic responsiveness.

What considerations are important when using IL13RA1 antibodies in cancer research?

When utilizing IL13RA1 antibodies in cancer research, several key considerations should guide experimental design and interpretation:

• Variable expression patterns: IL13RA1 expression varies significantly across cancer types and even within tumor subtypes. Comprehensive expression profiling using validated antibodies is essential before designing functional studies.

• IL13RA1/IL13RA2 discrimination: Cancer cells often express both IL13RA1 and IL13RA2. Given their structural similarity , antibodies must be carefully validated for specificity to avoid cross-reactivity that could confound results.

• Tissue context preservation: For studying IL13RA1 in the tumor microenvironment, methods preserving spatial relationships (immunohistochemistry, multiplex immunofluorescence) are preferable to dissociation-based approaches that disrupt tissue architecture.

• Post-translational modifications: Cancer-specific alterations in glycosylation or phosphorylation may affect antibody binding to IL13RA1. Validation in relevant cancer models is essential, with particular attention to phosphorylation sites like Tyr405 .

• Receptor trafficking dynamics: Cancer cells often display altered receptor trafficking. When studying IL13RA1 internalization or recycling, subcellular fractionation followed by western blotting or confocal microscopy with appropriate antibodies can map these processes.

• Tumor heterogeneity consideration: Single-cell analysis approaches combining IL13RA1 antibodies with other cancer markers can resolve heterogeneous expression patterns within tumors that might be masked in bulk analyses.

• Functional state assessment: Beyond mere expression, determining the functional state of IL13RA1 is critical. Phospho-specific antibodies targeting sites like Tyr405 can distinguish active from inactive receptor pools.

• Therapeutic targeting validation: For cancer immunotherapy approaches targeting IL13RA1, antibodies can validate target accessibility and expression before treatment and monitor receptor downregulation or masking during treatment.

• Cross-species considerations: When using animal models, account for the 76% amino acid sequence identity between human and mouse IL13RA1 when selecting antibodies for preclinical studies.

• Alternative splicing detection: Cancer cells may express splice variants of IL13RA1 with altered functional properties. Antibodies targeting different epitopes can help identify such variants.

• Prognostic biomarker potential: When evaluating IL13RA1 as a prognostic marker, standardized immunohistochemistry protocols with validated antibodies and scoring systems are essential for consistent assessment across patient cohorts.

How should researchers interpret variability in IL13RA1 detection across different assay systems?

When encountering variability in IL13RA1 detection across different assay systems, researchers should consider several factors to ensure proper data interpretation:

• Epitope accessibility differences: The epitope recognized by the IL13RA1 antibody may be differentially accessible in various assay formats. In native conditions (flow cytometry), certain epitopes may be exposed, while in denatured conditions (Western blot), different epitopes become accessible. The clone #419718 antibody used in flow cytometry applications recognizes the extracellular domain (Ala27-Thr343) , which may display different accessibility in various assay formats.

• Post-translational modifications: IL13RA1 undergoes various post-translational modifications that can affect antibody binding. Phosphorylation, particularly at sites like Tyr405 , glycosylation, and proteolytic processing may vary across sample preparation methods, leading to detection inconsistencies.

• Conformational dependencies: Some antibodies are conformation-dependent and perform differently in assays that preserve native structure (flow cytometry, immunoprecipitation) versus those that denature proteins (Western blotting). This is particularly relevant for antibodies targeting the complex extracellular domain of IL13RA1 .

• Sample preparation effects: Different lysis buffers, fixation protocols, or antigen retrieval methods can significantly impact epitope preservation and accessibility. Standardize these variables when comparing across studies.

• Expression level thresholds: Each detection method has a different sensitivity threshold. Flow cytometry using fluorophore-conjugated antibodies may detect lower IL13RA1 expression levels than colorimetric immunohistochemistry or Western blotting .

• Antibody performance characteristics: Consider antibody-specific characteristics like affinity, avidity, and potential cross-reactivity with related proteins such as IL13RA2 . These properties may manifest differently across assay platforms.

• Methodological analysis approach:

  • Implement titration studies across different platforms to determine optimal antibody concentrations for each

  • Use multiple antibody clones recognizing distinct epitopes to validate findings

  • Include appropriate positive controls (e.g., Myc-tagged IL13RA1-transfected 293T cells ) in each assay system

  • Validate findings with orthogonal methods (e.g., mRNA expression, reporter assays)

• Quantification method differences: Different quantification approaches (band intensity in Western blots, mean fluorescence intensity in flow cytometry, H-score in immunohistochemistry) each have unique dynamic ranges and limitations that affect data interpretation.

• Standardization approaches: To minimize variability, implement internal standards across experiments, use consistent protocols, and consider adopting community-established guidelines for specific applications.

What statistical approaches are recommended for analyzing IL13RA1 expression data across different experimental conditions?

When analyzing IL13RA1 expression data across experimental conditions, the following statistical approaches are recommended: