IL15RA Antibody

What is IL-15RA and why is it important in immunological research?

IL-15RA is the alpha subunit of the interleukin-15 receptor that specifically binds IL-15 with high affinity. IL-15 is a pleiotropic cytokine that plays crucial roles in both innate and adaptive immunity. It is mainly produced by activated monocytes, macrophages, and dendritic cells, and shares structural similarities with IL-2. Both cytokines utilize the same IL-2/15Rβ and common γ-c chain for signaling . IL-15RA is significant in research because it mediates the development of various effector cells, including natural killer (NK) cells and CD8 T cells, through a unique mechanism called trans-presentation . Understanding IL-15RA function is critical for developing therapies targeting immune-mediated diseases and cancer.

What are the key differences between commercial IL-15RA antibodies and how do I select the appropriate one for my experiment?

When selecting an IL-15RA antibody, researchers should consider several key factors:

Importantly, some antibodies target the interface where IL-15 binds to IL-15Rα, potentially rendering the receptor undetectable when IL-15 is bound . Therefore, selecting antibodies based on the specific research question and experimental design is crucial for obtaining reliable results.

What are the recommended dilutions and experimental conditions for using IL-15RA antibodies in different applications?

Based on validated protocols, here are the recommended conditions for different applications:

These recommendations serve as starting points, and titration in each specific experimental system is necessary for optimal results. Storage conditions (-20°C) and stability (typically one year after shipment) should be maintained for consistent performance .

How can I address inconsistent detection of IL-15RA in Western blot analyses?

Inconsistent detection of IL-15RA in Western blot analyses can arise from several factors:

• Molecular Weight Discrepancies: The observed molecular weight (55-65 kDa) differs significantly from the calculated weight (24 kDa) . This disparity likely results from post-translational modifications, particularly glycosylation.

• Sample Preparation Protocol:

  • Ensure complete denaturation with appropriate buffers containing SDS and reducing agents

  • Include protease inhibitors to prevent degradation

  • Consider using fresh samples, as freeze-thaw cycles may affect detection

• Antibody Selection Issues:

  • Some antibodies may recognize epitopes that become inaccessible when IL-15 is bound to IL-15RA

  • Use antibodies validated specifically for Western blot applications

• IL-15RA Isoform Specificity:

  • Multiple isoforms exist due to alternative splicing

  • Confirm which isoforms your antibody can detect based on the immunogen information

• Methodological Optimization:

  • Perform gradient dilution experiments (1:500-1:1000) to determine optimal concentration

  • Consider longer exposure times for weaker signals

  • Use positive controls (such as HeLa cells) known to express IL-15RA

The presence of different isoforms and post-translational modifications of IL-15RA contributes to detection challenges, requiring careful optimization of experimental protocols.

What strategies can improve detection sensitivity for IL-15RA in immunohistochemistry and immunofluorescence?

Improving detection sensitivity for IL-15RA in tissue sections requires attention to several critical factors:

• Optimal Antigen Retrieval:

  • For FFPE tissues, using TE buffer (pH 9.0) has shown better results for IL-15RA detection

  • Alternative approach: citrate buffer (pH 6.0) for tissues resistant to standard retrieval

• Antibody Dilution Optimization:

  • Start with the recommended range (1:50-1:500 for IHC)

  • Perform dilution series to identify optimal signal-to-background ratio

• Signal Amplification Methods:

  • Consider tyramide signal amplification for low-abundance targets

  • For immunofluorescence, use high-sensitivity detection systems

• Blocking Optimization:

  • Use 5% BSA in buffer for optimal blocking as indicated in antibody specifications

  • Extend blocking time (1-2 hours) to reduce background

• Tissue-Specific Considerations:

  • Positive controls: human liver cancer, spleen, kidney, heart, testis, skin, and brain tissues have demonstrated IL-15RA expression

  • Adjust protocols based on tissue-specific factors

• Multi-labeling Strategies:

  • For co-localization studies, carefully select primary antibodies from different host species

  • Use sequential detection protocols for challenging combinations

Thorough optimization of these parameters enables reliable detection of IL-15RA across different tissue types, which is essential for studying its distribution and expression patterns in normal and pathological conditions.

How can researchers effectively measure soluble IL-15RA in biological samples?

Measuring soluble IL-15RA (sIL-15RA) in biological samples presents significant challenges due to its complex biology:

• Detection Challenges:

  • The presence of multiple IL-15RA isoforms complicates quantification

  • Antibody epitope location critically affects detection - some antibodies target the IL-15 binding interface, making IL-15-bound IL-15RA undetectable

  • Some antibody combinations detect both free and IL-15-bound IL-15RA, preventing differentiation between forms

• Methodological Approaches:

  • ELISA: Select commercial kits with validated antibody pairs that specifically distinguish free vs. IL-15-bound forms

  • Flow Cytometry: Can detect sIL-15RA using bead-based multiplex assays

  • Western Blot: Useful for distinguishing different forms based on molecular weight differences

  • Mass Spectrometry: Consider for precise identification of specific isoforms

• Sample Processing Considerations:

  • Minimize freeze-thaw cycles

  • Process samples rapidly to prevent degradation

  • Standardize collection and storage protocols for comparative studies

• Control and Normalization Strategies:

  • Include recombinant sIL-15RA standards at known concentrations

  • Establish consistent detection thresholds across experiments

  • Document sample collection and storage conditions meticulously

Researchers should recognize that proteolytic cleavage of IL-15RA from cell surfaces is a physiologically relevant process that regulates IL-15 trans-presentation . Therefore, measured sIL-15RA levels reflect not only expression but also active regulatory mechanisms in the biological system being studied.

How does the IL-15 trans-presentation mechanism impact experimental design when studying IL-15RA function?

IL-15 trans-presentation is a unique cytokine delivery mechanism that significantly impacts experimental design:

• Mechanistic Understanding:

  • In trans-presentation, IL-15 bound to IL-15RA on presenting cells interacts with IL-2Rβ/γc on responding cells

  • This differs fundamentally from conventional cytokine-receptor interactions where all receptor components are on the responding cell

• Experimental Design Implications:

  • Cell Co-Culture Systems: Essential for studying natural trans-presentation

  • Co-culture models using IL-15Rα-expressing cells (e.g., HEK-293 cells) preloaded with IL-15 and responding cells (e.g., Kit225 cells) labeled with tracking dyes like VPD-450 enable direct observation of the process

  • Single-cell systems fail to recapitulate the intercellular dynamics

• Signaling Assessment:

  • Phosphorylated STAT5 (pSTAT5) serves as a primary readout for successful trans-presentation

  • Flow cytometry and AlphaScreen technology offer sensitive detection of pSTAT5

  • The duration of STAT5 signaling is affected by IL-15RA cleavage, requiring time-course experiments rather than single-timepoint measurements

• Detection Challenges:

  • Antibodies targeting the IL-15-binding interface of IL-15RA may fail to detect complexes

  • Differential labeling of presenting and responding cells is essential for distinguishing cell populations

• Cleavage Dynamics:

  • IL-15RA cleavage leads to the generation of soluble IL-15·IL-15RA complexes that affect responding cell behavior

  • Incorporating uncleavable IL-15RA mutants in experiments helps distinguish effects of membrane-bound versus cleaved complexes

Understanding these complex dynamics is essential for properly interpreting experimental results in IL-15/IL-15RA research, particularly when translating findings to therapeutic applications targeting this pathway.

What roles do different IL-15RA isoforms play in cytokine signaling, and how can researchers distinguish them experimentally?

IL-15RA exists in multiple isoforms with distinct biological functions, presenting significant experimental challenges:

• Isoform Diversity and Function:

  • Multiple isoforms arise from alternative splicing

  • Different isoforms exhibit varying affinities for IL-15

  • Isoforms may have distinct subcellular localizations and signaling properties

  • Some isoforms lack transmembrane domains and function as soluble receptors

• Experimental Approaches for Isoform Distinction:

• Functional Assessment Strategies:

  • Recombinant expression of individual isoforms in cell models

  • CRISPR/Cas9 engineering to selectively delete specific isoforms

  • Reporter assays to measure signaling outcomes of different isoforms

  • Subcellular localization studies using immunofluorescence with isoform-specific antibodies

• Analytical Considerations:

  • Include positive controls expressing known isoforms

  • Consider tissue-specific expression patterns of different isoforms

  • Account for post-translational modifications that affect molecular weight

Understanding isoform-specific functions is critical for developing targeted therapeutic approaches and interpreting experimental results in different biological contexts. The research community continues to develop tools for better discrimination between these functionally distinct molecular species.

How can researchers investigate the interplay between IL-15RA cleavage and downstream signaling events?

The proteolytic cleavage of IL-15RA represents a critical regulatory mechanism affecting IL-15 signaling that can be studied through several experimental approaches:

• Cleavage Mechanism Investigation:

  • Studies have shown that IL-15RA can be cleaved from presenting cells, generating soluble IL-15·IL-15RA complexes

  • Cleavage events are associated with changes in STAT5 phosphorylation duration in responding cells

  • Uncleavable IL-15RA mutants provide valuable tools for mechanistic studies

• Signaling Analysis Methods:

  • pSTAT5 Detection: Primary readout for IL-15 signaling using flow cytometry or AlphaScreen technology

  • Time-course Experiments: Essential for capturing the dynamics of signaling following cleavage events

  • Co-culture Systems: Required to study trans-presentation between presenting and responding cells

• Experimental Models:

  • Membrane-bound IL-15·IL-15Rα Fusion Proteins: Allow controlled study of presentation mechanics

  • Uncleavable IL-15Rα Mutants: Enable comparison between normal and prevented cleavage scenarios

  • IL-15Rα-expressing Cell Lines: HEK-293 cells expressing IL-15Rα can be preloaded with IL-15 for trans-presentation studies

• Key Experimental Observations:

  • IL-15 can be detected within responding cells following trans-presentation

  • Entry of IL-15 into responding cells requires cleavage of the IL-15·IL-15Rα complex

  • After separation from presenting cells, responding cells can recycle internalized IL-15·IL-15Rα complexes

  • This recycling supports residual proliferation for a limited time period

• Therapeutic Implications:

  • Understanding cleavage dynamics has led to the development of approaches like NANTIL-15, which efficiently reduced inflammation in disease models

  • Selective targeting of IL-15Rα can modulate inflammation through mechanisms distinct from complete IL-15 blockade

This complex interplay between receptor cleavage, complex internalization, and signaling represents an unprecedented cytokine pathway with significant implications for understanding IL-15 biology and developing targeted immunotherapeutics.

How should researchers interpret discrepancies between calculated and observed molecular weights of IL-15RA in Western blot analyses?

The significant difference between calculated (24 kDa) and observed (55-65 kDa) molecular weights of IL-15RA presents an important analytical challenge:

• Sources of Molecular Weight Discrepancies:

  • Post-translational Modifications: Extensive glycosylation is the primary cause

  • Protein Conformation: Non-fully denatured samples may migrate differently

  • Isoform Variation: Alternative splicing creates different molecular weight variants

  • Association with Other Proteins: Incomplete sample preparation may leave some protein complexes intact

• Analytical Approach:

  • Compare observed bands with positive controls (e.g., HeLa cells known to express IL-15RA)

  • Use glycosidase treatment to confirm glycosylation contribution to higher molecular weight

  • Consider running gradient gels to better resolve the protein bands

  • Include deglycosylation controls to establish baseline unmodified molecular weight

• Experimental Validation:

  • Knockout/knockdown controls confirm band specificity

  • Recombinant protein standards establish size reference points

  • Peptide competition assays verify antibody specificity

• Interpretation Guidelines:

• Documentation Recommendations:

  • Report both observed and expected molecular weights

  • Describe sample preparation methods in detail

  • Include positive and negative controls in publications

  • Acknowledge the known molecular weight discrepancy in interpretations

This characteristic molecular weight discrepancy is well-documented for IL-15RA and represents normal biological processing rather than experimental artifact when properly validated .

How can researchers distinguish between specific and non-specific binding when validating IL-15RA antibodies?

Distinguishing specific from non-specific binding is critical for generating reliable IL-15RA antibody data:

• Essential Validation Controls:

  • Positive Controls: Cell lines with confirmed IL-15RA expression (HeLa cells)

  • Negative Controls:

    • IL-15RA knockout cells (e.g., Kit225-15RαKO)

    • Secondary antibody-only controls

    • Isotype controls matching the primary antibody

  • Tissue Panels: Test across multiple tissues with known expression patterns (liver cancer, spleen, kidney, heart, testis, skin, brain)

• Critical Validation Experiments:

  • Peptide Competition Assays: Pre-incubation with immunizing peptide should eliminate specific signal

  • Antibody Dilution Series: Non-specific binding typically doesn't show dose-dependent reduction

  • Orthogonal Methods: Confirm findings using multiple detection techniques (WB, IHC, IF)

  • siRNA Knockdown: Demonstrate reduced signal with target knockdown

• Application-Specific Considerations:

• Advanced Validation Approaches:

  • Multiple antibodies targeting different epitopes should show concordant results

  • Recombinant expression systems with controlled IL-15RA levels

  • Cross-validation with genetic detection methods (RNA-seq, qPCR)

• Documentation Standards:

  • Report all validation experiments in publications

  • Include images of negative controls

  • Specify the exact epitope recognized by the antibody

  • Acknowledge any limitations in specificity

Thorough validation is essential as the quality of IL-15RA antibodies varies considerably between manufacturers and different lot numbers of the same product may show performance differences .

What are the best practices for analyzing IL-15RA expression in immune cell subpopulations using flow cytometry?

Flow cytometric analysis of IL-15RA expression in immune cell subpopulations requires careful optimization:

• Panel Design Considerations:

  • Include markers for definitive identification of relevant immune subpopulations

  • Select fluorophores with minimal spectral overlap for IL-15RA detection

  • Include viability dyes to exclude dead cells, which can bind antibodies non-specifically

• Sample Preparation Optimization:

  • Fresh samples yield more reliable results than frozen for surface IL-15RA detection

  • Standardize processing times as receptor expression may change ex vivo

  • For intracellular detection, use fixation and permeabilization reagents compatible with IL-15RA epitopes

• Staining Protocol Refinement:

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Include FcR blocking reagents to minimize non-specific binding

  • Consider sequential staining approaches for challenging markers

• Essential Controls:

  • Fluorescence Minus One (FMO) controls for accurate gating

  • Isotype controls matched to IL-15RA antibody

  • Biological controls (stimulated vs. unstimulated cells)

  • IL-15RA knockout or knockdown samples when available

• Advanced Analytical Approaches:

  • Trans-presentation Studies: Label Kit225 cells with tracking dyes like VPD-450 when co-cultured with IL-15Rα-expressing cells to distinguish cell populations

  • Signaling Analysis: Incorporate phospho-STAT5 detection for functional assessment

  • Receptor Internalization: Time-course experiments to track receptor dynamics

  • Multi-parametric Analysis: Use dimensionality reduction techniques (tSNE, UMAP) for complex datasets

• Data Interpretation Guidelines:

  • Report both percentage of positive cells and median fluorescence intensity

  • Consider both membrane and intracellular expression patterns

  • Account for potential masking of epitopes when IL-15 is bound to IL-15RA

  • Correlate flow cytometry findings with functional assays when possible

This comprehensive approach enables accurate assessment of IL-15RA expression across different immune cell populations, critical for understanding its role in immune regulation and potential therapeutic targeting.

How can researchers leverage IL-15RA antibodies to study the effects of targeted therapeutics on the IL-15 signaling pathway?

IL-15RA antibodies serve as powerful tools for evaluating therapeutic interventions targeting the IL-15 pathway:

• Therapeutic Mechanism Assessment:

  • Receptor Occupancy Analysis: Determine if therapeutics block IL-15 binding to IL-15RA

  • Pathway Inhibition Measurement: Quantify changes in downstream STAT5 phosphorylation

  • Trans-presentation Interference: Assess disruption of IL-15·IL-15RA complex formation or presentation

• Molecular Targeting Approaches:

  • Recent research demonstrates that selective targeting of IL-15Rα (e.g., NANTIL-15) can reduce inflammation

  • This approach differs from complete IL-15 blockade by retaining IL-15Rα binding while preventing IL-2Rβ recruitment

  • Interface-specific antibodies can distinguish these mechanisms

• Experimental Models for Therapeutic Evaluation:

  • Cell-Based Assays: pSTAT5 signaling in Kit225 cells provides quantitative readout for inhibitor efficacy

  • Co-Culture Systems: Essential for evaluating trans-presentation disruption

  • In Vivo Models: Inflammation models (e.g., collagen-induced arthritis) demonstrate therapeutic effects

• Biomarker Development:

  • Changes in soluble IL-15RA levels may serve as pharmacodynamic markers

  • Receptor occupancy on specific immune cell subsets can indicate therapeutic engagement

  • IL-15RA antibodies enable monitoring of these parameters in clinical samples

• Combination Therapy Assessment:

  • Analyze changes in IL-15RA expression/function when combined with other immunomodulatory agents

  • Evaluate potential synergistic or antagonistic effects on the IL-15 pathway

  • Determine optimal sequencing of therapeutic interventions

These applications highlight how IL-15RA antibodies not only serve as research tools but also enable translational studies critical for therapeutic development targeting this important immunoregulatory pathway.

What are the current limitations in IL-15RA antibody technology, and how might these be addressed in future research?

Current IL-15RA antibody technologies face several limitations that impact research outcomes:

• Epitope Accessibility Challenges:

  • Some antibodies target the IL-15/IL-15Rα interface, making detection impossible when IL-15 is bound

  • This creates blind spots in detecting physiologically relevant complexes

  • Future Direction: Development of conformation-specific antibodies that recognize the complex rather than individual components

• Isoform Discrimination Limitations:

  • Current antibodies often cannot distinguish between multiple IL-15RA isoforms

  • This obscures isoform-specific functions in different biological contexts

  • Future Direction: Generation of isoform-specific antibodies targeting unique junction sequences

• Species Cross-Reactivity Issues:

  • Many antibodies show limited cross-reactivity between human and model organisms

  • This complicates translation between preclinical models and human studies

  • Future Direction: Development of multi-species reactive antibodies or matched antibody panels

• Complex Formation Analysis:

  • Limited tools exist for specifically detecting IL-15·IL-15Rα complexes in their native state

  • Future Direction: Proximity ligation assays and split complementation technologies

• Quantification Standardization:

  • Lack of standardized reference materials for absolute quantification

  • Variability between antibody lots affects reproducibility

  • Future Direction: Development of certified reference materials and standardized protocols

• Technological Innovations on the Horizon:

  • Single-molecule imaging of IL-15/IL-15RA interactions

  • Aptamer-based detection alternatives to antibodies

  • CRISPR-engineered reporter systems for pathway activation

  • Nanobody and single-domain antibody alternatives for improved tissue penetration

Addressing these limitations will require collaborative efforts between academic researchers and commercial antibody developers to create next-generation reagents that enable more precise understanding of IL-15RA biology in health and disease.

How can researchers integrate IL-15RA antibody-based assays with other omics technologies for comprehensive pathway analysis?

Integrating IL-15RA antibody data with multi-omics approaches enables systems-level understanding of this signaling pathway:

• Integrated Multi-Omics Strategies:

• Advanced Experimental Approaches:

  • Cellular Indexing of Transcriptomes and Epitopes (CITE-seq): Simultaneously profile IL-15RA protein expression and transcriptomes in single cells

  • CO-Detection by indEXing (CODEX): Multiplex imaging of IL-15RA alongside dozens of other proteins in tissue contexts

  • Spatial Transcriptomics: Correlate IL-15RA protein localization with local transcriptional environments

• Computational Integration Methods:

  • Network analysis algorithms to identify IL-15RA-centered regulatory hubs

  • Machine learning approaches to predict IL-15RA expression patterns from multi-omics data

  • Causal inference models to establish directional relationships in signaling networks

• Biological Systems for Integrated Analysis:

  • IL-15 Trans-presentation Models: Extend beyond protein detection to multi-omics profiles of presenting and responding cells

  • Inflammation Models: Connect IL-15RA expression patterns to global changes in diseased tissues

  • Therapeutic Response Studies: Use multi-omics to comprehensively profile pathway modulation

• Practical Implementation Strategies:

  • Sequential processing of samples for compatible multi-omics analyses

  • Parallel analysis of replicate samples with different technologies

  • Use of common reference standards across platforms

  • Integrated database solutions for multi-omics data storage and retrieval

This integrated approach transforms isolated antibody-based observations into comprehensive understanding of IL-15RA's role within the complex immune signaling network, potentially revealing new therapeutic targets and biomarkers.