IL13RA1 Antibody, Biotin conjugated

What is IL13RA1 and what is its biological significance?

IL13RA1 functions as a primary subunit of the interleukin 13 receptor complex. It plays a crucial role in binding interleukin-13 (IL13) with low affinity and, when combined with the IL4 receptor alpha (IL4RA), forms a functional receptor for IL13 signaling. Beyond its role in IL13 signaling, IL13RA1 also serves as an alternate accessory protein to the common cytokine receptor gamma chain for interleukin-4 (IL4) signaling pathways . The receptor has significant involvement in mediating signaling processes that activate important downstream molecules including JAK1, STAT3, and STAT6, which are induced by both IL13 and IL4 . This signaling cascade is critical for various immunological responses, particularly in allergic inflammation and immune responses to parasitic infections.

What species reactivity should be expected with commercially available IL13RA1 biotin-conjugated antibodies?

Commercial IL13RA1 biotin-conjugated antibodies typically demonstrate specific reactivity profiles based on the immunogen used in their production. Most commonly, these antibodies are raised against human IL13RA1 protein and show strong reactivity with human samples . Some antibody products exhibit broader reactivity, with manufacturers reporting cross-reactivity with mouse and rat samples . When selecting an antibody for experimental use, it is essential to verify the documented species reactivity provided by the manufacturer. For example, some commercially available polyclonal antibodies are specifically raised against human IL13RA1 (residues 22-234AA) and have confirmed human reactivity , while others may be produced using immunogens from different species or regions of the protein. Experimental validation through positive controls from the species of interest is always recommended before proceeding with full-scale experiments.

What are the recommended dilutions and applications for IL13RA1 biotin-conjugated antibodies?

IL13RA1 biotin-conjugated antibodies can be employed across several research applications with specific dilution recommendations for optimal results. For Western blotting (WB), the typical dilution range is 0.5-2 μg/ml, which provides sufficient sensitivity while minimizing background . Immunohistochemistry (IHC) applications generally require higher antibody concentrations, with recommended dilutions of 5-20 μg/ml to ensure adequate tissue penetration and specific antigen detection . For immunofluorescence or immunocytochemistry (IF/ICC), similar concentrations of 5-20 μg/ml are recommended . ELISA applications typically use dilutions ranging from 1:500 to 1:5000, depending on the specific assay format and detection sensitivity requirements . These recommended dilutions serve as starting points, and researchers should conduct optimization experiments to determine the ideal concentration for their specific experimental conditions, sample types, and detection systems.

What are the optimal storage and handling conditions for maintaining IL13RA1 biotin-conjugated antibody activity?

To maintain optimal activity of IL13RA1 biotin-conjugated antibodies, proper storage and handling are essential. These antibodies are typically supplied in liquid form, often in a buffer containing PBS (pH 7.4) with preservatives like Proclin-300 and stabilizers such as glycerol (50%) . For long-term storage, manufacturers recommend aliquoting the antibody and storing at -20°C or -80°C to minimize freeze-thaw cycles, which can degrade antibody activity . Repeated freeze-thaw cycles should be strictly avoided as each cycle can significantly reduce antibody potency. When working with the antibody, it should be thawed gradually at refrigerated temperatures (4°C) rather than at room temperature to preserve structural integrity. For daily use, small working aliquots can be maintained at 4°C for up to one week, but extended storage at this temperature is not recommended. Before using the antibody, gentle mixing by inversion (not vortexing) helps ensure homogeneity without causing protein denaturation or aggregation.

How should experimental controls be designed when using IL13RA1 biotin-conjugated antibodies?

Robust experimental design with appropriate controls is critical when using IL13RA1 biotin-conjugated antibodies. Primary negative controls should include isotype controls (rabbit IgG-biotin for polyclonal antibodies) at equivalent concentrations to verify that observed signals are not due to non-specific binding of immunoglobulins . Additionally, secondary reagent-only controls (using streptavidin-conjugated detection reagents without primary antibody) help identify potential background from the detection system. Positive controls should include samples with known IL13RA1 expression levels, such as cell lines that express IL13RA1 (e.g., certain immune or epithelial cell lines). For antibody validation, blocking peptide controls can be valuable - pre-incubating the antibody with excess recombinant IL13RA1 protein should abolish specific staining in immunohistochemistry or reduce band intensity in Western blotting. When performing functional studies on IL13RA1 signaling, parallel experiments with IL13RA1 knockdown or knockout models provide additional validation. For mechanistic studies, including JAK/STAT pathway inhibitors can help confirm specificity of observed signaling effects.

How can biotin interference be mitigated in experiments using biotin-conjugated IL13RA1 antibodies?

Biotin interference represents a significant technical challenge when using biotin-conjugated antibodies in certain experimental systems. This interference occurs when endogenous biotin or biotin supplements in biological samples compete with biotin-conjugated antibodies for binding to streptavidin detection reagents . To mitigate this issue, researchers can implement several strategies: First, perform a biotin blocking step by pre-incubating samples with free streptavidin to sequester endogenous biotin before adding biotin-conjugated antibodies. Second, utilize commercially available biotin blocking kits specifically designed to neutralize endogenous biotin. Third, consider sample preparation methods that minimize biotin content, such as dialysis or specific extraction protocols. For cell culture experiments, using biotin-free media for 24-48 hours prior to sample collection can significantly reduce background. When working with tissue samples from subjects with potential biotin supplementation, additional washing steps and extended blocking protocols may be necessary. In cases where biotin interference persists, alternative detection methods using non-biotin conjugated primary antibodies should be considered .

What strategies can optimize signal-to-noise ratio in immunohistochemistry and immunofluorescence with IL13RA1 biotin-conjugated antibodies?

Achieving optimal signal-to-noise ratios with IL13RA1 biotin-conjugated antibodies requires methodological refinement across several experimental parameters. Begin with thorough optimization of antibody concentration through titration experiments; while the recommended range is 5-20 μg/ml for IHC and IF/ICC , the optimal concentration may vary between tissue types and fixation methods. Implement extended blocking steps (30-60 minutes) using blocking buffers containing both protein blockers (BSA, normal serum) and detergents (0.1-0.3% Triton X-100 or Tween-20) to reduce non-specific binding. Antigen retrieval methods should be systematically tested, including heat-induced epitope retrieval with citrate or EDTA buffers at varying pH levels to maximize target epitope accessibility. Incorporate additional washing steps between antibody incubations, using buffers containing 0.05-0.1% Tween-20 to remove unbound antibodies more effectively. Consider signal amplification systems such as tyramide signal amplification (TSA) for detecting low-abundance IL13RA1, but be aware this may increase background if not properly optimized. Finally, counterstain optimization is critical - reduce counterstain concentration if it masks specific signals, and utilize spectral imaging or linear unmixing in multi-color immunofluorescence to resolve overlapping emission spectra.

How can specificity of IL13RA1 detection be confirmed in experimental systems?

Confirming the specificity of IL13RA1 detection requires a multi-faceted validation approach. First, employ molecular validation by using siRNA or shRNA knockdown of IL13RA1 in relevant cell lines, which should result in reduced signal intensity proportional to the knockdown efficiency. Second, perform absorption controls by pre-incubating the biotin-conjugated IL13RA1 antibody with recombinant IL13RA1 protein (such as the 22-234AA immunogen region mentioned in product specifications) prior to application; this should significantly diminish specific staining. Third, compare staining patterns across multiple IL13RA1 antibodies targeting different epitopes, as consistent localization patterns provide strong evidence for specificity. Fourth, conduct parallel analyses using orthogonal techniques - correlation between protein detection methods (IHC, Western blot) and mRNA expression (qPCR, in situ hybridization) strengthens confidence in antibody specificity. For functional validation, demonstrate that detected IL13RA1 participates in expected signaling events by showing co-localization with downstream effectors like phosphorylated STAT6 after IL-13 stimulation. Finally, include both positive control tissues/cells with known IL13RA1 expression and negative controls where IL13RA1 is absent or minimally expressed to establish the dynamic range of detection.

How can IL13RA1 biotin-conjugated antibodies be utilized to investigate JAK/STAT signaling pathways?

IL13RA1 biotin-conjugated antibodies provide valuable tools for dissecting the complex JAK/STAT signaling pathways activated downstream of IL-13 binding. The IL13RA1 receptor has been demonstrated to bind tyrosine kinase TYK2 and mediate signaling processes leading to the activation of JAK1, STAT3, and STAT6 . To investigate these pathways, researchers can employ co-immunoprecipitation experiments using biotin-conjugated IL13RA1 antibodies followed by streptavidin pull-down and subsequent immunoblotting for JAK1, TYK2, or STAT proteins to examine physical associations between the receptor and these signaling molecules. Proximity ligation assays (PLA) represent another advanced application, where biotin-conjugated IL13RA1 antibodies paired with antibodies against phosphorylated JAK/STAT components can visualize active signaling complexes at the single-molecule level within cells. For temporal studies of pathway activation, stimulate cells with IL-13 for various time intervals before fixation and dual immunofluorescence staining with IL13RA1 biotin-conjugated antibodies and phospho-specific antibodies against JAK1, STAT3, or STAT6, allowing quantification of co-localization and trafficking dynamics. Flow cytometry applications can simultaneously measure cell surface IL13RA1 levels and intracellular phosphorylated STAT proteins in heterogeneous cell populations, providing insights into signaling heterogeneity among different cell subsets.

What methodological approaches can differentiate between IL13RA1 and IL13RA2 receptor expression and function?

Distinguishing between IL13RA1 and IL13RA2 expression and function requires sophisticated methodological approaches due to their distinct but related roles in IL-13 biology. IL13RA2 acts as a high-affinity decoy for IL13 that mediates internalization and depletion of extracellular IL13, whereas IL13RA1 forms functional signaling complexes with IL4RA . For differential expression analysis, use multicolor flow cytometry with distinctly labeled antibodies against each receptor subtype to quantify their relative abundance on cell surfaces. Implement dual immunofluorescence microscopy with subtype-specific antibodies to visualize their spatial distribution and potential co-localization. For functional differentiation, conduct parallel siRNA knockdown experiments targeting each receptor individually, followed by assessment of IL-13-induced STAT6 phosphorylation - knockdown of IL13RA1 should substantially reduce signaling, while IL13RA2 knockdown might enhance signaling by increasing IL-13 availability. Reporter gene assays using STAT6-responsive elements can further quantify these functional differences. Receptor internalization dynamics can be monitored through antibody-based pulse-chase experiments, where differential trafficking patterns between IL13RA1 (recycling for continued signaling) and IL13RA2 (predominantly degradative) can be observed. For mechanistic studies, generate chimeric receptors containing the extracellular domains of one receptor subtype fused to the intracellular domain of the other to dissect domain-specific functions in signal transduction.

How can IL13RA1 biotin-conjugated antibodies contribute to investigating the role of IL-13 signaling in allergic inflammation and immune responses?

IL13RA1 biotin-conjugated antibodies enable sophisticated investigation of IL-13 signaling in allergic inflammation and immune responses through multiple experimental approaches. IL-13 plays critical roles in allergic inflammation and immune responses to parasite infection, synergizing with IL-2 in regulating interferon-gamma synthesis and stimulating B-cell proliferation and activation of eosinophils, basophils, and mast cells . For tissue-based studies, perform multiplex immunohistochemistry or immunofluorescence using biotin-conjugated IL13RA1 antibodies in combination with cell-type specific markers and phospho-STAT6 to identify which cell populations actively respond to IL-13 in inflamed tissues. Laser capture microdissection of IL13RA1-positive cells followed by transcriptomic analysis can reveal cell-type specific gene expression signatures downstream of IL-13 signaling. In functional blocking studies, compare the effects of anti-IL13 antibodies versus anti-IL13RA1 antibodies on allergic responses in experimental models to distinguish receptor-specific effects from ligand neutralization. For investigating the antagonistic relationship between Th1 and Th2 responses, use IL13RA1 detection in combination with T-bet (Th1) and GATA3 (Th2) transcription factor staining to visualize the polarization status of T-cell populations. To explore IL13RA1's role in regulating other inflammatory pathways, conduct co-immunoprecipitation studies to identify novel interaction partners that might mediate its suppressive effects on NF-κB signaling and proinflammatory cytokine production. Finally, examine the relationship between IL13RA1 expression and VCAM1 induction in endothelial cells to elucidate mechanisms of eosinophil recruitment in allergic inflammation .

What are the critical parameters for optimizing Western blot protocols using IL13RA1 biotin-conjugated antibodies?

Optimizing Western blot protocols for IL13RA1 detection with biotin-conjugated antibodies requires careful attention to several critical parameters. Sample preparation should include protease inhibitors to preserve the integrity of IL13RA1 protein, which has a predicted molecular weight of approximately 49 kDa . For electrophoresis, gradient gels (4-12% or 4-15%) often provide better resolution for membrane proteins like IL13RA1. Transfer conditions should be optimized for membrane proteins - using lower methanol concentrations (10% instead of 20%) in the transfer buffer can improve transfer efficiency of hydrophobic proteins. When blocking, BSA (3-5%) may be preferred over milk as milk contains biotin that could interfere with the biotin-streptavidin detection system . The recommended antibody dilution range of 0.5-2 μg/ml should be tested systematically to determine optimal concentration . Incubation with the biotin-conjugated primary antibody should be performed at 4°C overnight to maximize specific binding while minimizing background. For detection, high-sensitivity streptavidin-HRP conjugates should be used at manufacturer-recommended dilutions, typically 1:2000-1:5000. Extended washing steps (5 washes of 5 minutes each) with TBS-T (0.1% Tween) after both primary and secondary reagent incubations significantly improve signal-to-noise ratio. When visualizing, incrementally adjusted exposure times help identify the optimal detection window before signal saturation occurs.

How should researchers design experiments to study IL13RA1 receptor internalization and trafficking?

Studying IL13RA1 receptor internalization and trafficking requires sophisticated experimental designs that track receptor dynamics following ligand binding. Begin with live-cell imaging approaches using biotin-conjugated IL13RA1 antibodies paired with fluorescently-labeled streptavidin to label surface receptors, followed by the addition of IL-13 to trigger internalization. Time-lapse confocal microscopy can then capture the temporal dynamics of receptor endocytosis. For greater specificity, biotinylated IL-13 ligand can be used instead of antibodies to visualize only the binding-competent receptor fraction. Pulse-chase experiments offer quantitative measurements of internalization rates - surface receptors are labeled with biotin-conjugated antibodies, allowed to internalize for varying time points following IL-13 stimulation, then remaining surface biotin is stripped, and internalized biotin-labeled receptors are quantified. Co-localization studies with markers for different endocytic compartments (early endosomes: EEA1; recycling endosomes: Rab11; late endosomes: Rab7; lysosomes: LAMP1) can map the trafficking route of internalized IL13RA1. For mechanistic studies, implement pharmacological inhibitors of different endocytic pathways (dynamin inhibitors, clathrin inhibitors, etc.) to determine the molecular machinery required for IL13RA1 internalization. Flow cytometry provides quantitative measurement of surface versus internalized receptors across cell populations, while biochemical approaches like surface biotinylation followed by immunoprecipitation can distinguish between surface and internal receptor pools at different time points after stimulation.