IL13RA2 Antibody

What is IL13RA2 and why is it an important research target?

IL13RA2 (interleukin 13 receptor subunit alpha 2) is a cell surface receptor protein involved in immune response and inflammatory processes. It is also known by several alternative names including CD213a2, CT19, IL-13R, IL13BP, IL-13 receptor subunit alpha-2, and IL-13R subunit alpha-2 . This protein has a molecular mass of approximately 44.2 kilodaltons and plays a critical role in modulating immune responses . IL13RA2 has gained significant research interest due to its implications in various diseases, particularly cancer and allergic conditions, making it both a valuable biomarker and potential therapeutic target . The receptor's ability to regulate inflammation and immune function positions it as a key focus for immunological research and drug development targeting dysregulated immune responses .

Which tissue types express IL13RA2 and how does expression vary in pathological conditions?

IL13RA2 expression has been detected in multiple human tissues, with notable presence in placenta and testis as demonstrated through Western blot analysis using specific antibodies . Additionally, immunohistochemistry studies have revealed significant IL13RA2 expression in prostate cancer tissue, specifically localized to glandular epithelial cells . Expression levels can be modified by inflammatory cytokines, with research showing that TNF (tumor necrosis factor) can induce upregulation of IL13RA2 in certain cell types, as documented in flow cytometry experiments comparing unstimulated and cytokine-stimulated cells . Several studies have also demonstrated that IL13RA2 expression is significantly elevated in various cancer types, making it a valuable oncological research target and a potential cancer biomarker . The expression pattern observed in pathological versus normal tissue provides researchers with important insights into the receptor's role in disease progression.

What is the functional relationship between IL13RA2 and the IL-13 signaling pathway?

IL13RA2 functions as a regulator of both IL-13 and IL-4 signal transduction, as demonstrated in studies using primary human fibroblasts . Research has shown that IL13RA2 can modulate the biological activity of IL-13 variants, such as R110Q, suggesting its importance in controlling IL-13-mediated immune responses . The level of IL13RA2 expression directly impacts receptor distribution and IL-13 signaling efficiency, indicating a dose-dependent regulatory mechanism . Importantly, IL13RA2 appears to serve as a decoy receptor in some contexts, potentially sequestering IL-13 cytokine and preventing its interaction with the signaling IL-13 receptor complex. This regulatory function has significant implications for understanding inflammatory and allergic conditions where IL-13 plays a central role . Manipulation of this pathway through targeted antibodies or recombinant proteins offers potential therapeutic approaches for treating IL-13-mediated pathologies.

How should researchers select the appropriate IL13RA2 antibody for their specific application?

When selecting an IL13RA2 antibody, researchers should prioritize the intended application and species reactivity requirements. The search results indicate over 599 IL13RA2 antibodies are commercially available across 32 suppliers, with varying application validations . For Western blot applications, antibodies validated specifically for this technique, such as the Aviva Systems Biology IL13RA2 antibody targeting the middle region, would be appropriate choices . For multipurpose research, antibodies validated across multiple applications, such as the R&D Systems Human IL-13 R alpha 2 Antibody (which is validated for WB, FCM, ICC, IHC, IHC-p, IP, MC, and neutralization assays) may provide greater experimental flexibility . Researchers should also consider the specific epitope recognition, as some antibodies target particular regions of the protein that may be more accessible depending on experimental conditions. Additionally, review the published literature where specific antibodies have been used successfully for your application of interest, and examine available validation data such as Western blot images or flow cytometry profiles provided by manufacturers .

What validation steps should be performed before using an IL13RA2 antibody in critical experiments?

Before employing an IL13RA2 antibody in critical experiments, researchers should conduct a series of validation steps to confirm specificity and functionality. Begin with Western blot analysis using positive control samples known to express IL13RA2, such as placenta or testis tissue lysates, which have been documented to show specific bands at approximately 50-55 kDa under reducing conditions . For flow cytometry applications, validation should include comparison of staining between cells with confirmed IL13RA2 expression (such as cytokine-stimulated cells) versus unstimulated controls or cells known not to express the target . A critical validation step involves performing competition assays using soluble recombinant IL13RA2 to confirm binding specificity, as demonstrated in published research where pre-incubation with soluble receptor competed for antibody binding . For immunohistochemistry applications, researchers should validate staining patterns against known expression profiles, such as the glandular epithelial cell staining observed in prostate cancer tissue . Additionally, validation should include negative controls such as isotype-matched control antibodies to rule out non-specific binding, and when possible, genetic approaches like knockdown/knockout systems or cells engineered to express IL13RA2 should be employed as definitive controls .

What are the known cross-reactivity issues with IL13RA2 antibodies and how can they be addressed?

Cross-reactivity issues have been documented with certain commercially available IL13RA2 antibodies, particularly the B-D13 antibody, which has been shown to contain two distinct monoclonal antibodies that may recognize different antigens . Research has demonstrated that some commercial preparations may recognize not only IL13RA2 but potentially other proteins like VCAM-1, leading to misinterpretation of results . To address these issues, researchers should perform soluble receptor competition studies, where pre-incubation of the antibody with recombinant IL13RA2 should significantly reduce staining if the antibody is truly specific . Additionally, mass spectrometry analysis of antibody preparations can reveal whether they contain multiple antibody species, as demonstrated in studies showing distinct mass species for both heavy and light chains in certain commercial preparations . Researchers should also validate specificity through multiple techniques (e.g., if using an antibody for flow cytometry, confirm expression patterns with Western blot or qPCR) . When possible, utilize genetically modified cells with controlled expression of IL13RA2 as definitive positive and negative controls to establish antibody specificity . Finally, comparing multiple antibodies from different sources or those recognizing different epitopes can provide convergent evidence of specific recognition.

What are the optimal conditions for detecting IL13RA2 by Western blot?

For optimal Western blot detection of IL13RA2, researchers should follow several key methodological considerations based on published protocols. Use PVDF membrane for protein transfer, as documented in successful detection of IL13RA2 in human placenta and testis tissue lysates . Employ reducing conditions with Immunoblot Buffer Group 1, which has been shown to effectively resolve IL13RA2 bands . The expected molecular weight of IL13RA2 under these conditions appears as a specific band at approximately 50-55 kDa, though variations between 55-59 kDa have been reported depending on the specific sample preparation and detection system used . For primary antibody incubation, a concentration of 0.2 μg/mL of anti-IL13RA2 antibody has proven effective, followed by appropriate HRP-conjugated secondary antibodies (such as Anti-Goat IgG HRP conjugate when using goat polyclonal primary antibodies) . For loading controls, a protein concentration of 0.2 mg/mL of tissue lysate has been successfully employed in published studies . Simple Western™ automated western blotting systems can also be used for more standardized results, with documented success using 10 μg/mL of anti-IL13RA2 antibody and a 12-230 kDa separation system . Researchers should particularly note that sample preparation methods may affect the apparent molecular weight, with variations between conventional Western blot (50-55 kDa) and Simple Western™ systems (approximately 59 kDa) .

How can IL13RA2 expression be accurately quantified in different cell types?

Accurate quantification of IL13RA2 expression in different cell types requires a multi-method approach. At the mRNA level, quantitative PCR (qPCR) has been successfully employed to measure IL13RA2 transcripts in various cell lines including U251T, PBT003-4, PBT008, and THP-1 cells, with results normalized to multiple housekeeping genes (ACTB, UBC, GAPDH, and RPLP0) for reliable quantification . For protein-level quantification, flow cytometry provides a powerful tool for measuring IL13RA2 surface expression, as demonstrated in studies comparing expression levels between different cell lines and under various treatment conditions . When performing flow cytometry, researchers should include appropriate isotype controls and consider using geometric mean fluorescence intensity (MFI) ratios for standardized comparison across experiments . Western blot analysis with densitometry can provide semi-quantitative measurement of total cellular IL13RA2 protein levels, while ELISA-based methods can offer more precise quantification when standardized against known concentrations of recombinant protein . For cellular localization and expression heterogeneity within tissues, immunohistochemistry or immunocytochemistry should be employed, with quantification possible through digital image analysis of staining intensity and distribution patterns . Importantly, researchers should be aware that IL13RA2 expression can be dynamically regulated by cytokines such as TNF, IL-4, and IL-13, requiring careful experimental design when comparing expression levels under different conditions .

What methods are recommended for studying IL13RA2 in flow cytometry experiments?

For flow cytometry analysis of IL13RA2, researchers should follow several methodological recommendations based on published protocols. Begin by optimizing cell preparation, using gentle dissociation methods that preserve surface epitopes, especially for adherent cell lines like U251T cells that express IL13RA2 . Careful attention should be paid to antibody selection, as validation studies have revealed significant differences in specificity between commercial antibodies, with the R&D Systems AF146 antibody demonstrating reliable detection of IL13RA2 in flow cytometry applications . When staining, use appropriate blocking steps to minimize non-specific binding, and include relevant isotype control antibodies matched to the primary antibody species, class, and conjugation . For stimulation experiments, cytokine treatments (such as TNF combined with either IL-4 or IL-13) have been shown to effectively induce IL13RA2 expression in certain cell types, providing useful positive controls . To confirm specificity of detection, competition assays using pre-incubation with soluble recombinant IL13RA2-Fc are highly recommended, as this approach has successfully distinguished specific binding from cross-reactivity in published research . When quantifying expression, report relative fluorescence index (RFI) values compared to unstimulated controls to standardize results across experiments . For mechanistic studies, consider incorporating transcription blockers (Actinomycin D) or translation inhibitors (Cycloheximide) at titrated concentrations (Actinomycin D: 0.06-0.3 μg/ml; Cycloheximide: 2.5-10 μg/ml) to investigate regulation of IL13RA2 expression, as these approaches have provided valuable insights into receptor regulation .

How does IL13RA2 expression affect IL-13 signaling and what are the implications for targeting this pathway?

IL13RA2 expression levels directly impact IL-13 signaling dynamics and have significant implications for therapeutic targeting of this pathway. Research has demonstrated that IL13RA2 functions as a key regulator of both IL-13 and IL-4 signal transduction in primary human fibroblasts . Specifically, the expression level of IL13RA2 impacts receptor distribution throughout the cell and consequently affects IL-13 signaling efficiency . Studies investigating the IL-13 variant R110Q have shown that IL13RA2 levels modulate the biological activity of this variant, suggesting potential implications for personalized medicine approaches that consider receptor expression profiles . The mechanistic understanding of IL13RA2's regulatory role has advanced through experiments using neutralizing antibodies, which have revealed that blocking IL13RA2 can enhance IL-13-mediated effects in certain cellular contexts . This dual role of IL13RA2—functioning both as a decoy receptor that sequesters IL-13 and as a potential signaling molecule under specific conditions—creates complex considerations for therapeutic intervention. For developing targeted therapies, researchers must assess whether inhibiting or enhancing IL13RA2 function would be more beneficial, depending on the disease context and the specific role the receptor plays in pathology . These nuanced relationships between receptor expression and signaling outcomes provide critical insights for drug development strategies targeting the IL-13 pathway in conditions ranging from allergic inflammation to cancer.

What are the current challenges in developing neutralizing antibodies against IL13RA2?

Developing effective neutralizing antibodies against IL13RA2 presents several significant challenges that researchers continue to address. A primary difficulty involves ensuring antibody specificity, as studies have revealed that some commercially available antibodies purported to target IL13RA2 may recognize multiple antigens, including unrelated proteins like VCAM-1 . This cross-reactivity issue has been documented through soluble receptor competition studies and mass spectrometry analysis of antibody preparations, highlighting the need for rigorous validation procedures . Another challenge concerns the dynamic regulation of IL13RA2 expression, which can be rapidly induced by cytokine stimulation (e.g., TNF combined with IL-4 or IL-13) and is dependent on both transcription and translation, as demonstrated in studies using Actinomycin D and Cycloheximide . This expression variability complicates target accessibility across different tissue types and disease states. Furthermore, the development of antibodies with optimal neutralizing capacity requires detailed understanding of the receptor's functional domains and binding interfaces, information that continues to evolve through structural and functional studies . For therapeutic applications, researchers must also address challenges related to antibody penetration into tissues where IL13RA2 is expressed pathologically, such as in solid tumors. Additionally, the potential for compensatory mechanisms following IL13RA2 neutralization, including upregulation of alternative signaling pathways, represents a significant consideration for therapeutic development . Addressing these challenges requires integrated approaches combining advanced antibody engineering, comprehensive validation strategies, and thorough understanding of IL13RA2 biology in both normal and pathological contexts.

How should researchers interpret conflicting data from different IL13RA2 antibodies?

When facing conflicting data from different IL13RA2 antibodies, researchers should implement a systematic analytical approach. First, examine the epitope specificity of each antibody, as antibodies targeting different regions of IL13RA2 may yield varying results depending on protein conformation, post-translational modifications, or interactions with other molecules . Publication evidence has revealed that some commercial B-D13 antibody preparations contain mixtures of antibodies with different specificities, potentially explaining contradictory results . Researchers should verify antibody composition through techniques like mass spectrometry, which has successfully identified distinct mass species for both heavy and light chains in certain commercial preparations . For resolving conflicts, perform cross-validation using multiple detection methods; for example, if flow cytometry and Western blot yield different results, incorporate qPCR to determine whether protein expression correlates with transcript levels . Competition assays using soluble recombinant IL13RA2 can definitively test specificity, as demonstrated in studies where pre-incubation with soluble receptor reduced binding of truly specific antibodies . When evaluating published literature or comparing results between laboratories, consider differences in experimental conditions, cell types, and cytokine stimulation protocols, as IL13RA2 expression is highly regulated and context-dependent . Finally, for critical experiments, researchers should ideally utilize multiple validated antibodies targeting different epitopes and confirm findings using genetic approaches such as knockout/knockdown models or overexpression systems .

What factors might contribute to variability in IL13RA2 detection across experiments?

Multiple factors can contribute to variability in IL13RA2 detection across experiments, requiring careful consideration during experimental design and data interpretation. Cell culture conditions significantly impact IL13RA2 expression, with research demonstrating that cytokine stimulation (specifically TNF combined with either IL-4 or IL-13) can dramatically upregulate receptor levels in certain cell types . The timing of analysis is crucial, as IL13RA2 induction is dependent on both transcription and translation, with experiments using Actinomycin D and Cycloheximide revealing the kinetics of this regulation . Technical variations in antibody quality between lots or suppliers can introduce significant inconsistency, as documented in studies comparing different lots of PE-conjugated B-D13 antibody that showed varying detection efficacy . Sample preparation methods also impact results, with observed differences in apparent molecular weight between conventional Western blot (50-55 kDa) and Simple Western™ systems (approximately 59 kDa) . For flow cytometry, variations in cell fixation, permeabilization, and staining protocols can affect epitope accessibility and antibody binding efficiency . Post-translational modifications of IL13RA2 may vary across cell types or disease states, potentially masking or exposing different epitopes recognized by various antibodies . Finally, differences in detection sensitivity between methods (for example, enhanced chemiluminescence versus fluorescence-based Western blot detection) can result in apparent discrepancies when measuring low-abundance IL13RA2 expression . Acknowledging and controlling for these variables is essential for generating reproducible data in IL13RA2 research.

How is IL13RA2 being investigated as a biomarker in cancer and inflammatory diseases?

IL13RA2 is emerging as a significant biomarker candidate across multiple disease contexts. In oncology, immunohistochemical studies have demonstrated elevated IL13RA2 expression in prostate cancer tissue, specifically localized to glandular epithelial cells, suggesting potential diagnostic applications . Research has expanded to investigate IL13RA2 expression across various tumor types, with particular focus on gliomas where the receptor has shown promise as both a biomarker and therapeutic target . The development of IL13RA2-integrated genetically engineered mouse models has further advanced research into its role in pediatric high-grade gliomas, providing platforms for both biomarker validation and therapeutic testing . Beyond oncology, IL13RA2's role in regulating IL-13 and IL-4 signal transduction positions it as a potential biomarker for inflammatory and allergic conditions where these cytokines play central roles . Studies examining IL13RA2 in combination with other markers may provide more comprehensive disease signatures, enhancing diagnostic accuracy and prognostic value. Current research is also exploring whether soluble forms of IL13RA2 in biological fluids could serve as minimally invasive biomarkers, potentially allowing for liquid biopsy approaches in disease monitoring . As analytical technologies advance, multiplexed detection systems incorporating IL13RA2 alongside other disease-relevant markers offer promising approaches for personalized medicine applications, potentially guiding treatment decisions based on receptor expression profiles.

What recent technological advances have improved IL13RA2 detection and characterization?

Recent technological advances have significantly enhanced the detection and characterization of IL13RA2 across multiple research applications. The development of highly specific antibodies with validated performance across various applications (Western blot, flow cytometry, immunohistochemistry) has improved detection reliability and cross-platform consistency . Automated Western blotting systems, such as the Simple Western™ technology referenced in the search results, offer increased standardization and quantitative precision for protein expression analysis, with documented protocols successfully detecting IL13RA2 at approximately 59 kDa . Advanced flow cytometry protocols have evolved to include multiparameter analysis capabilities, allowing researchers to simultaneously assess IL13RA2 expression alongside other markers and cellular characteristics . Mass spectrometry-based approaches have proven valuable not only for antibody validation (identifying multiple antibody species in commercial preparations) but also for detailed characterization of the IL13RA2 protein itself, including post-translational modifications and interaction partners . Genetic engineering techniques, including the development of cell lines and mouse models with controlled IL13RA2 expression, provide powerful tools for studying receptor function in defined contexts . High-throughput transcriptomic methods enable researchers to examine IL13RA2 expression patterns across diverse cell types and disease states, with normalization to multiple housekeeping genes (ACTB, UBC, GAPDH, RPLP0) enhancing quantitative reliability . Finally, advanced imaging techniques, including multiplexed immunofluorescence approaches, allow for detailed analysis of IL13RA2 distribution within tissues and co-localization with other proteins of interest, providing spatial context to expression data .