IL2RA Recombinant Monoclonal Antibody

What is IL2RA and what is its biological significance?

IL2RA, also known as CD25, interleukin-2 receptor subunit alpha, or p55, is a critical component of the interleukin-2 receptor complex. In humans, this protein consists of 272 amino acid residues with a molecular mass of approximately 30.8 kDa . IL2RA is localized in the cell membrane and plays a fundamental role in the regulation of immune tolerance by controlling regulatory T cell (Treg) activity. Specifically, IL2RA is involved in suppressing the activation and expansion of autoreactive T cells, making it essential for maintaining self-tolerance and preventing autoimmunity .

The receptor for IL-2 exists in three forms with varying affinities for IL-2: low-affinity (homodimeric IL2RA chains), medium-affinity (homodimeric IL2RB chains), and high-affinity receptors (heterotrimeric complexes containing IL2RA, IL2RB, and IL2RG) . This structural versatility allows for differential responses to IL-2 signaling depending on the cellular context and activation state.

Which cell types and tissues express IL2RA?

IL2RA demonstrates a specific expression pattern across various tissues and cell types. It is notably expressed in the tonsil, spleen, lymph node, and cerebellum . At the cellular level, IL2RA serves as a marker for identifying several important immune cell populations, including:

• Large Intestine Lamina Propria Lymphocytes

• T Follicular Regulatory Cells

• Regulatory T Cells (Tregs)

• T Regulatory Cells

Understanding this expression pattern is crucial for researchers designing experiments involving immune cell identification and functional characterization. IL2RA expression is particularly important in studies of T cell development, activation, and regulatory function.

What research applications are available for IL2RA antibodies?

IL2RA recombinant monoclonal antibodies serve multiple research applications, with specific antibody products validated for different techniques. Based on the available data, these antibodies can be effectively used in:

• Western Blotting (WB)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Flow Cytometry

• Immunoprecipitation (IP)

• Immunohistochemistry on paraffin-embedded tissues (IHC-p)

• Immunohistochemistry on frozen sections (IHC-fr)

When selecting an IL2RA antibody for specific applications, researchers should verify that the particular clone has been validated for their intended use. For example, the BLR157J clone has been confirmed effective for ICC, Flow Cytometry, IP, and WB applications with human samples .

How can IL2RA antibodies be used to investigate regulatory T cell development?

IL2RA antibodies play a crucial role in studying the developmental stages and functional maturation of regulatory T cells (Tregs). Research has demonstrated that IL-2R signaling is required by thymic Tregs at two distinct developmental stages: an early step for expansion and survival, and a later step for functional maturation .

To effectively study these processes, researchers can employ IL2RA antibodies in flow cytometry experiments to identify and isolate Tregs at various developmental stages. This approach allows for detailed analysis of how IL-2 signaling influences Treg development in both thymic and peripheral compartments. When using tamoxifen-inducible CD25 knockout models, investigators can temporally control IL2RA expression to distinguish between IL-2-mediated effects in the thymus versus those in peripheral tissues .

Methodologically, researchers should consider the following protocol elements:

• Use of PHA (5 μg/mL for 48 hours) to stimulate PBMCs before IL2RA antibody staining

• Co-staining with anti-CD3 antibodies to identify T cell populations

• Selection of appropriate secondary antibodies (e.g., APC-conjugated Anti-Human IgG)

What methods can validate the specificity of IL2RA recombinant monoclonal antibodies?

Validating antibody specificity is critical for obtaining reliable research results. For IL2RA recombinant monoclonal antibodies, multiple validation approaches should be employed:

• Functional blocking assays: Testing the antibody's ability to block IL-2 binding to IL2RA-expressing cells. For example, using biotinylated IL-2 protein (500 ng/mL) to bind HEK293 cells transfected with recombinant human CD25/IL-2R alpha, then demonstrating that the binding is completely blocked by 0.5 μg/mL of human anti-human CD25/IL-2R alpha monoclonal antibody .

• Immunoprecipitation validation: Using the antibody to immunoprecipitate IL2RA from cellular lysates (e.g., 0.5 mg per IP reaction from MJ cells prepared using NETN lysis buffer), then confirming the identity of the precipitated protein by Western blot using a different antibody against a non-overlapping epitope of IL2RA .

• Comparison with known positive and negative controls: Including appropriate isotype controls (e.g., normal human IgG) in parallel experiments to confirm specificity .

• Cross-validation with multiple detection methods: Confirming antibody specificity across different applications (WB, flow cytometry, IHC) to ensure consistent target recognition.

What are the optimal protocols for flow cytometry using IL2RA antibodies?

Flow cytometry is one of the most common applications for IL2RA antibodies, particularly for identifying and characterizing regulatory T cells. Based on the research literature, the following methodological considerations are important:

• Cell preparation and stimulation:

  • For optimal detection of IL2RA expression, human PBMCs should be treated with PHA (5 μg/mL) for 48 hours prior to staining

  • Fresh isolation of cells is preferred to avoid loss of surface marker expression

• Staining protocol:

  • Primary antibody: Use IL2RA/CD25 recombinant monoclonal antibody at manufacturer-recommended concentration (typically 0.5-1 μg/mL)

  • Secondary detection: APC-conjugated anti-human IgG secondary antibody if using unconjugated primary antibodies

  • Co-staining: Include CD3 PE-conjugated monoclonal antibody to identify T cell populations

• Controls:

  • Include isotype control antibodies (e.g., normal human IgG) at the same concentration as the primary antibody

  • Include unstained cells and single-color controls for compensation

• Analysis considerations:

  • Gate on lymphocyte population based on forward and side scatter properties

  • Analyze CD3+ cells for IL2RA expression to identify regulatory T cell populations

  • Consider additional markers (FoxP3, CD127) for more definitive Treg identification

How do IL2RA signaling pathways influence T cell functional outcomes?

IL2RA is a critical component of the high-affinity IL-2 receptor complex, and its signaling mechanisms significantly impact T cell function. The IL2RA receptor complex consists of three chains: IL2RA (CD25), IL2RB, and IL2RG. The gamma chain is also shared by interleukin 4 and interleukin 7 receptors .

Functionally, IL2RA signaling influences multiple T cell outcomes:

• Growth and proliferation: IL-2 signaling through the high-affinity receptor promotes T cell expansion, with IL2RA expression being critical for this process.

• Survival regulation: IL2RA-dependent signaling is required for thymic Treg expansion and survival .

• Functional maturation: Beyond survival, IL2RA signaling is necessary for the functional maturation of Tregs, enabling their suppressive activity .

• Prevention of autoimmunity: The regulatory role of IL2RA is demonstrated by the development of ulcerative colitis-like disease in mice with targeted disruption of the gene, suggesting its importance in immune responses to antigenic stimuli .

For effective investigation of these pathways, researchers can use IL2RA antibodies to block receptor function or to identify cells with active IL-2 signaling. Genetic approaches using inducible knockout models can temporally control IL2RA expression to distinguish between its roles in development versus maintenance of T cell function .

What are the key considerations for immunoprecipitation using IL2RA antibodies?

Immunoprecipitation (IP) is a valuable technique for studying protein-protein interactions and post-translational modifications of IL2RA. Based on validated protocols, researchers should consider the following methodological details:

• Lysate preparation:

  • Use NETN lysis buffer for optimal protein extraction

  • Prepare whole cell lysate at concentration of 0.5 mg per IP reaction

• Antibody amounts:

  • Use approximately 20 μL of antibody per mg of lysate for efficient immunoprecipitation

  • Load 5% of IP product for subsequent Western blot analysis

• Validation approach:

  • Use a second antibody targeting a different epitope of IL2RA for Western blot detection

  • This cross-validation ensures specificity of the immunoprecipitated protein

• Controls:

  • Include isotype control antibodies to identify non-specific binding

  • Consider using lysates from cells not expressing IL2RA as negative controls

• Detection sensitivity:

  • Use chemiluminescence detection for optimal sensitivity when blotting immunoprecipitated IL2RA

How should researchers select between different IL2RA antibody formats?

IL2RA antibodies are available in multiple formats, including unconjugated and directly conjugated versions (APC, FITC, biotin, HRP, Alexa fluor). The selection of appropriate format depends on the specific research application:

• Unconjugated antibodies:

  • Ideal for Western blotting, ELISA, and applications requiring secondary antibody amplification

  • Offer flexibility for custom conjugation to preferred fluorophores or enzymes

  • Required for IP applications where the antibody needs to bind protein A/G

• Fluorophore-conjugated antibodies:

  • Optimal for flow cytometry and immunofluorescence applications

  • APC-conjugated antibodies provide excellent sensitivity for flow cytometry

  • FITC-conjugated antibodies work well for immunohistochemistry on paraffin sections

• Enzyme-conjugated antibodies:

  • HRP-conjugated formats are ideal for direct detection in Western blots and ELISA

  • Eliminate the need for secondary antibody incubation, shortening protocols

• Consideration for multicolor analysis:

  • When designing multicolor flow cytometry panels, select IL2RA antibody conjugates that complement other fluorophores in the panel

  • Ensure minimal spectral overlap between fluorophores to reduce compensation requirements

Each antibody format has specific advantages depending on the experimental design, sensitivity requirements, and multiplexing needs of the research application.

What are the reconstitution and storage recommendations for IL2RA antibodies?

Proper handling of IL2RA antibodies is essential for maintaining their performance and specificity. Based on manufacturer recommendations:

• Reconstitution guidelines:

  • Add 100 μL distilled water to achieve a final antibody concentration of approximately 1 mg/mL

  • For carrier-free antibodies intended for conjugation experiments, perform an additional round of desalting using appropriate columns (e.g., Zeba Spin Desalting Columns, 7KMWCO)

• Working concentration ranges:

  • Flow cytometry: 0.5-1 μg/mL

  • Immunoprecipitation: 20 μL/mg of lysate

  • Western blotting: 1:1000 dilution for detection of immunoprecipitated proteins

• Storage recommendations:

  • Store reconstituted antibodies at 4°C for short-term use (1-2 weeks)

  • For long-term storage, prepare aliquots and store at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles that can degrade antibody performance

• Quality control measures:

  • Perform regular validation tests to ensure antibody performance does not degrade over time

  • Include appropriate positive and negative controls in each experiment

How do IL2RA mutations impact immune function and disease development?

Mutations in the IL2RA gene have significant implications for immune function and are associated with several pathological conditions. Research using IL2RA antibodies has helped elucidate these relationships:

• Interleukin 2 receptor alpha deficiency:

  • Mutations in IL2RA are directly associated with this immunodeficiency condition

  • Characterized by impaired T cell function and immune dysregulation

• Autoimmune manifestations:

  • The targeted disruption of the IL2RA gene in mice leads to ulcerative colitis-like disease, demonstrating its role in preventing autoimmunity

  • This model suggests IL2RA's importance in immune responses to antigenic stimuli

• Regulatory T cell dysfunction:

  • IL2RA is crucial for the regulation of immune tolerance by controlling regulatory T cell activity

  • Mutations affecting IL2RA expression or function impair the suppression of autoreactive T cells

• Research model considerations:

  • Tamoxifen-inducible CD25 knockout models allow temporal control of IL2RA expression to distinguish between developmental and maintenance roles

  • Germline versus Treg-targeted mutations in the IL2RA gene provide insights into cell-specific functions

Researchers investigating these conditions should consider using IL2RA antibodies for phenotyping affected tissues, characterizing T cell populations, and evaluating therapeutic interventions targeting the IL-2 pathway.

What role does IL2RA play in thymic versus peripheral Treg development?

IL2RA (CD25) has distinct roles in thymic development versus peripheral maintenance of regulatory T cells. Understanding these differences is crucial for research on Treg biology:

• Thymic Treg development:

  • IL-2R signaling is required at an early developmental stage for expansion and survival of thymic Tregs

  • A second, distinct IL-2R signaling event is needed later for functional maturation of these cells

• Methodological approaches to distinguish developmental stages:

  • Tamoxifen-inducible CD25 knockout models allow researchers to separate IL-2-mediated effects within the thymus from those in peripheral tissues

  • This model enables the temporal control of IL2RA expression to study stage-specific requirements

• Experimental design considerations:

  • Use CD25 wild-type littermates as experimental controls for all knockout strains

  • For histological analysis of inflammation in tissue sections, blind the identities of mouse strains

  • Choose sample sizes based on power calculations from preliminary results and published phenotypic data

• Research applications:

  • IL2RA antibodies can be used to identify and isolate Tregs at different developmental stages

  • Flow cytometry protocols using IL2RA antibodies allow quantification of Treg populations in thymus versus peripheral lymphoid tissues

These research approaches enable detailed investigation of how IL-2 signaling influences distinct aspects of Treg biology throughout development and maintenance.

How can researchers overcome common challenges when using IL2RA antibodies?

Working with IL2RA antibodies may present several technical challenges. Here are methodological approaches to address common issues:

• Low signal in flow cytometry:

  • Pre-stimulate cells with PHA (5 μg/mL for 48 hours) to upregulate IL2RA expression before staining

  • Use APC-conjugated secondary antibodies for enhanced sensitivity

  • Ensure fresh isolation of cells to prevent receptor internalization or shedding

• Non-specific binding in immunohistochemistry:

  • Implement thorough blocking steps using appropriate sera or protein blockers

  • Include isotype control antibodies at the same concentration as the primary antibody

  • Optimize antibody concentration through titration experiments

• Inconsistent immunoprecipitation results:

  • Use NETN lysis buffer for optimal protein extraction

  • Maintain proper antibody-to-lysate ratio (approximately 20 μL antibody per mg of lysate)

  • Confirm results by Western blotting with a different antibody against a non-overlapping epitope

• Reduced antibody performance over time:

  • Store antibodies according to manufacturer recommendations

  • Prepare small aliquots to avoid repeated freeze-thaw cycles

  • For carrier-free antibodies intended for conjugation, perform additional desalting to remove stabilizers

By implementing these methodological refinements, researchers can optimize experimental protocols and obtain more consistent, reliable results when working with IL2RA antibodies.