IL2RG Antibody

What is IL2RG and why is it important in immunological research?

IL2RG (Interleukin 2 Receptor Gamma) is a critical component of multiple cytokine receptor complexes, functioning as the common gamma chain (γc) in receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The protein consists of 369 amino acid residues with a molecular weight of approximately 42.3 kDa, and is primarily localized to the cell membrane . IL2RG is essential for immune cell development and function, making it a crucial target for immunological research. The receptor is notably expressed in tissues including the stomach, spleen, rectum, gallbladder, and colon . Researchers target IL2RG to understand cytokine-mediated signaling pathways and immune response mechanisms, particularly in the context of primary immunodeficiencies, as mutations in IL2RG cause X-linked severe combined immunodeficiency (SCID). The IL2RG marker is also valuable for identifying Lympho-Myeloid Progenitor (Lmpp) Cells, making antibodies against this protein important tools for cellular immunology .

Which applications are most suitable for IL2RG antibodies?

IL2RG antibodies are versatile reagents applicable to multiple immunological techniques. Western blotting (WB) is particularly effective for detecting denatured IL2RG protein samples, with most commercially available antibodies optimized for this application . Immunohistochemistry (IHC) is suitable for visualizing IL2RG expression in both paraffin-embedded (IHC-p) and frozen tissue sections (IHC-f) . Immunocytochemistry (ICC) and immunofluorescence (IF) techniques allow researchers to examine cellular distribution patterns of IL2RG . Additionally, immunoprecipitation (IP) and enzyme-linked immunosorbent assay (ELISA) provide complementary approaches for protein interaction studies and quantitative measurements, respectively . When selecting an antibody for a specific application, researchers should verify the validated applications listed by manufacturers and consider antibody format (polyclonal vs. monoclonal) based on experimental needs. Optimization of dilution factors is essential, as noted in product literature stating "optimal dilutions should be determined by the end user" .

How do researchers select the appropriate IL2RG antibody for cross-species studies?

When conducting cross-species studies, researchers must carefully evaluate antibody reactivity profiles. Many IL2RG antibodies demonstrate confirmed reactivity with human, mouse, and rat IL2RG proteins . For studies involving other species, researchers should consult prediction scores based on sequence homology. For example, some antibodies show predicted reactivity with pig, bovine, sheep, and dog IL2RG proteins . These predictions are typically based on the alignment of immunogen sequences, with higher scores (>80) indicating greater confidence for successful detection . When selecting antibodies for novel species applications, researchers should perform validation studies using positive controls from the target species. It's also advisable to compare the amino acid sequence of the immunogen with the orthologous sequence in the target species. The IL2RG protein sequence is relatively conserved across mammalian species, with orthologs reported in mouse, rat, bovine, frog, chimpanzee, and chicken species , but sequence variations may affect epitope recognition.

How can IL2RG antibodies be used to investigate cytokine receptor complex formation?

Investigating cytokine receptor complex formation with IL2RG antibodies requires sophisticated experimental approaches. Researchers can employ co-immunoprecipitation (co-IP) assays using anti-IL2RG antibodies to pull down receptor complexes, followed by western blotting to detect associated receptor subunits. This approach can reveal the dynamics of heterodimeric and heterotrimeric receptor assembly. For more detailed structural insights, proximity ligation assays (PLA) can be conducted using pairs of antibodies targeting IL2RG and other receptor components (IL2RA, IL2RB, IL7R, etc.) to visualize receptor interactions in situ with subcellular resolution .

Advanced research has demonstrated the potential of bispecific antibodies targeting both IL-2Rβ and IL-2Rγ subunits to mimic IL-2 activity by inducing receptor dimerization and signaling . This approach "combines the favorable drug-like properties of antibodies with the functional behavior of a molecule that facilitates IL-2Rβ and IL-2Rγ association and downstream signaling" . Researchers investigating receptor complex formation should consider using complementary antibodies against different epitopes of IL2RG to avoid interference with binding sites involved in receptor-receptor or receptor-ligand interactions. Flow cytometry with multiple fluorescently-labeled antibodies can provide quantitative analysis of receptor complex formation on different immune cell subsets.

What are the best methodological approaches for studying IL2RG-mediated signaling using antibodies?

Studying IL2RG-mediated signaling demands multifaceted experimental designs using strategic antibody applications. Phospho-specific flow cytometry represents a powerful approach, where cells are stimulated with relevant cytokines (IL-2, IL-7, IL-15), fixed, permeabilized, and then stained with antibodies detecting phosphorylated signaling molecules (pSTAT5, pAKT, pERK) alongside anti-IL2RG antibodies to correlate receptor expression with signaling activation . For examining the functional consequences of receptor engagement, researchers can employ antibody-mediated receptor clustering using anti-IL2RG antibodies, either immobilized on plates or in solution with secondary cross-linking antibodies.

The development of bispecific IL2Rβγ antibodies has provided novel tools for studying receptor signaling, as these constructs "simultaneously bind IL-2Rβ and IL-2Rγ subunits and therefore mimic the activity of IL-2 while avoiding binding to IL-2Rα" . Researchers have observed that these bispecific antibodies exhibit "a range of agonist activity" with some demonstrating "agonist activity at a similar level to that seen with rhIL-2 in immune effector cells measured by phosphorylation of STAT5" . When designing signaling experiments, researchers should carefully time sample collection after stimulation, as signaling events occur in cascades with distinct temporal profiles. Inhibitor studies using JAK inhibitors in conjunction with IL2RG antibody detection can help delineate specific signaling pathways downstream of receptor activation.

How can researchers effectively use IL2RG antibodies to differentiate immune cell subsets?

Differentiating immune cell subsets using IL2RG antibodies requires sophisticated multiparameter analysis. The most effective approach combines anti-IL2RG antibodies with additional lineage markers in multicolor flow cytometry panels. When designing such panels, researchers should select anti-IL2RG antibodies with bright fluorochromes (PE, APC) if the expected expression is moderate to low, and consider the spectral overlap with other markers in the panel. IL2RG expression varies between immune cell populations, making it valuable for identifying specific subsets when used in conjunction with other markers .

For identifying Lympho-Myeloid Progenitor (Lmpp) Cells, researchers can employ IL2RG in combination with additional hematopoietic stem and progenitor cell markers . Mass cytometry (CyTOF) provides an alternative approach that allows simultaneous detection of dozens of markers, including IL2RG, without concerns about spectral overlap. When performing imaging studies, immunofluorescence microscopy using anti-IL2RG antibodies alongside lineage-specific markers can reveal the spatial distribution of receptor expression across different cell types within tissues. Researchers should validate sorting strategies based on IL2RG expression by confirming the identity and functionality of sorted populations through additional phenotypic and functional assays.

What are common challenges when using IL2RG antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with IL2RG antibodies. One frequent issue is variable staining intensity due to receptor internalization following cytokine stimulation. To address this, researchers should standardize the timing between stimulation and staining, or consider using fixation methods that capture cell surface proteins prior to stimulation. Another challenge is distinguishing specific from non-specific binding in tissues with high background. This can be mitigated by using appropriate blocking reagents (5-10% serum from the same species as the secondary antibody), including isotype controls, and validating staining patterns with multiple antibodies targeting different epitopes of IL2RG .

Post-translational modifications, particularly glycosylation, can affect antibody recognition of IL2RG . For applications sensitive to these modifications, researchers should select antibodies validated for detecting the relevant form of the protein. When analyzing data, it's important to account for the potential presence of two different isoforms of IL2RG that have been reported . Researchers experiencing weak or absent signals should systematically optimize antibody concentration, incubation conditions, and antigen retrieval methods (for IHC). The manufacturer guidance that "optimal dilutions should be determined by the end user" underscores the importance of titration experiments for each specific application and sample type .

How can researchers validate IL2RG antibody specificity in their experimental systems?

Validating IL2RG antibody specificity is crucial for generating reliable data. A comprehensive validation approach should include multiple complementary strategies. Positive controls using cell lines with known high expression of IL2RG (such as activated T cells or NK cells) can confirm antibody functionality. Negative controls should include cell lines lacking IL2RG expression or tissues from IL2RG knockout animals when available. For applications like western blotting, researchers should verify that the detected band appears at the expected molecular weight of approximately 42 kDa .

Knockdown validation using siRNA or CRISPR-Cas9 targeting IL2RG provides compelling evidence of antibody specificity, as the signal should decrease proportionally to the reduction in target protein. Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide, can demonstrate binding specificity, as the peptide should block antibody binding to the target protein. When using polyclonal antibodies like the rabbit polyclonal antibody referenced in the search results , researchers should be particularly thorough in validation due to the potential for batch-to-batch variation. Cross-validation using antibodies from different suppliers or those targeting different epitopes can provide additional confidence in the observed patterns of IL2RG expression.

How should researchers interpret contradictory results from different IL2RG antibody-based assays?

When faced with contradictory results from different IL2RG antibody-based assays, researchers should systematically evaluate several factors that might explain the discrepancies. First, consider epitope accessibility differences between assays—epitopes may be masked in certain applications due to protein conformation, fixation effects, or protein-protein interactions. Second, evaluate the impact of detection methods, as direct fluorescent conjugates may yield different results compared to secondary antibody detection systems. Different applications expose antibodies to varying conditions that might affect binding affinity and specificity .

The biological context is equally important—IL2RG expression and localization can change dramatically depending on cell activation state, differentiation status, and cytokine environment. Contradictory results might reflect actual biological differences rather than technical artifacts. When integrating data across platforms, researchers should preferentially trust results from properly validated antibodies and those that align with orthogonal measures of IL2RG expression (such as mRNA levels). For critical research findings, confirmation with multiple antibodies and complementary non-antibody-based methods (such as gene reporter assays) is recommended. Finally, researchers should consider that the two reported isoforms of IL2RG might be differentially detected by various antibodies, potentially explaining seemingly contradictory results .

How are IL2RG antibodies being utilized in the development of novel immunotherapies?

IL2RG antibodies are advancing immunotherapy development through multiple innovative applications. One cutting-edge approach involves the creation of bispecific antibodies that simultaneously target IL-2Rβ and IL-2Rγ subunits, activating IL-2 receptor signaling without preferentially stimulating regulatory T cells (Tregs) . Researchers have developed "a panel of fully human bispecific antibodies that simultaneously bind IL-2Rβ and IL-2Rγ subunits and therefore mimic the activity of IL-2 while avoiding binding to IL-2Rα" . This strategy has demonstrated promising results, as these bispecific antibodies "exhibit the desired activation and expansion of immune effector cells, while also avoiding preferential expansion of suppressive T-regs both in vitro and in vivo" .

The development pathway for these therapeutics involved using "next generation sequencing (NGS)-based antibody discovery platform using humanized rats (UniRats) to identify a large collection of binding domains with a wide range of agonist activity" . For researchers pursuing similar work, methodological considerations include the design of bispecific constructs—one successful approach utilized "knobs-into-holes technology to facilitate heavy-chain heterodimer formation, with a single anti-IL-2Rγ VH on the knob arm and a single anti-IL-2Rβ VH on the hole arm" . The therapeutic potential of these approaches is underscored by toxicity studies showing the bispecific agonist antibodies are "well tolerated in non-human primates with no observation of vascular leak syndrome or other toxicities" , addressing a key limitation of IL-2 based immunotherapies.

What role do IL2RG antibodies play in studying primary immunodeficiencies?

IL2RG antibodies serve as essential tools for studying primary immunodeficiencies, particularly X-linked severe combined immunodeficiency (X-SCID) caused by mutations in the IL2RG gene. Researchers utilize these antibodies to characterize patient samples, assess protein expression levels, and investigate the functional consequences of specific mutations. Flow cytometry with anti-IL2RG antibodies enables quantification of receptor expression on different immune cell populations from patients and carriers, providing insights into genotype-phenotype correlations. Immunofluorescence microscopy can reveal abnormal subcellular localization of mutant IL2RG proteins that might retain partial functionality.

For mechanistic studies, IL2RG antibodies facilitate the examination of downstream signaling defects in patient-derived cells, particularly through phospho-flow approaches detecting STAT5 phosphorylation following cytokine stimulation. In gene therapy development for X-SCID, these antibodies are crucial for monitoring IL2RG expression in transduced cells and assessing restoration of receptor complexes and signaling pathways. Additionally, IL2RG antibodies can be employed in immunoprecipitation studies to investigate whether specific mutations disrupt protein-protein interactions within receptor complexes. Researchers studying hypomorphic IL2RG mutations associated with atypical presentations of immunodeficiency particularly benefit from quantitative analyses using calibrated flow cytometry with anti-IL2RG antibodies to correlate expression levels with clinical phenotypes.

How can advanced microscopy techniques leverage IL2RG antibodies for spatial analysis of cytokine signaling?

Advanced microscopy techniques combined with IL2RG antibodies enable sophisticated spatial analysis of cytokine signaling microenvironments. Super-resolution microscopy methods such as Stimulated Emission Depletion (STED) and Single-Molecule Localization Microscopy (SMLM) using fluorescently-labeled anti-IL2RG antibodies can reveal nanoscale receptor clustering patterns that correlate with signaling efficiency. For dynamic studies, researchers can implement live-cell imaging with minimally disruptive anti-IL2RG antibody fragments (Fabs) or nanobodies to track receptor movement following cytokine engagement or immune synapse formation.

Multiplexed imaging techniques like Imaging Mass Cytometry (IMC) or Multiplexed Ion Beam Imaging (MIBI) allow simultaneous visualization of IL2RG alongside dozens of other markers in tissue sections, providing unprecedented insights into the spatial organization of cytokine signaling niches. For studying receptor interactions in situ, Förster Resonance Energy Transfer (FRET) microscopy using appropriately labeled antibody pairs targeting IL2RG and other receptor components can detect molecular proximity with nanometer resolution. When designing advanced microscopy experiments, researchers should carefully select antibodies with demonstrated specificity in imaging applications and consider using site-specific labeling approaches to avoid interfering with receptor function. These methodologies are particularly valuable for understanding how the spatial organization of IL2RG-containing receptor complexes influences signaling outcomes in complex tissues like lymphoid organs, inflammatory sites, or tumor microenvironments.