IL10RA Antibody

What is IL10RA and what is its biological function?

IL10RA (also known as IL-10R1 or CD210) is the alpha subunit of the interleukin-10 receptor complex. It functions as part of a heterotetramer comprising two IL10Rα molecules and two IL10Rβ molecules. IL10RA serves as the primary ligand-binding component of this receptor complex . The IL-10 signaling pathway plays a critical role in maintaining immune system balance through anti-inflammatory functions. IL10RA is expressed on most hematopoietic cells at basal levels but becomes upregulated upon cell activation, indicating its importance in inhibitory immune pathways . The protein is encoded by the IL10RA gene located on chromosome 11 in humans .

What is the expression pattern of IL10RA across different cell types?

IL10RA expression varies significantly by cell type and activation state:

|Cell Type|Basal Expression|Upon Activation|
|--|--|
|Naïve CD4+ T cells|Low|Upregulated after in vivo anti-CD3 treatment, particularly in Th17 cells in small intestine|
|Memory/effector T cells|Low|Upregulated following stimulation|
|Regulatory T cells|Low|Upregulated following stimulation|
|Human neutrophils|Low|Upregulated after LPS or IL4 stimulation|
|Dendritic cells|Present|Downregulated after maturation, enabling enhanced proinflammatory response|
|Fibroblasts|Low/Absent|Can be induced with LPS stimulation|
|Colonic epithelial cells|Constitutively expressed|Maintained expression|
|Macrophages|Present|Expression influences differentiation into MHCII low or MHCII high phenotypes|

This expression pattern highlights IL10RA's dynamic regulation in the immune system, particularly at mucosal surfaces .

What are the typical molecular characteristics of IL10RA protein?

IL10RA has the following molecular properties:

The discrepancy between calculated and observed molecular weights (63 kDa vs. 70-95 kDa) is likely due to post-translational modifications such as glycosylation .

What types of IL10RA antibodies are available for research?

Researchers have access to several types of IL10RA antibodies with different characteristics:

When selecting an antibody, researchers should consider the specific experimental application, species of interest, and validation data provided by manufacturers .

How should researchers determine the appropriate dilution for IL10RA antibody experiments?

Appropriate dilution depends on the specific application and antibody concentration. For commercially available antibodies such as Proteintech 13356-1-AP, the following dilutions are recommended:

These are starting points, and optimal dilutions should be determined empirically for each experimental system. It is recommended that antibodies be titrated in each testing system to obtain optimal results, as dilution requirements may be sample-dependent .

What are the validated applications for IL10RA antibodies?

IL10RA antibodies have been validated for multiple experimental applications:

Each application requires specific optimization and controls for reliable results.

What are the recommended protocols for IL10RA detection by Western blot?

For optimal detection of IL10RA by Western blot:

• Sample preparation: Prepare cell/tissue lysates in appropriate lysis buffer containing protease inhibitors

• Protein separation: Load 20-50 μg protein per lane on 10-12% SDS-PAGE gels

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Incubate with IL10RA antibody at 1:500-1:2000 dilution overnight at 4°C

• Washing: Wash membrane 3-5 times with TBST

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody

• Detection: Visualize using ECL substrate

Expected molecular weights for IL10RA are typically 70-72 kDa and 90-95 kDa, which differ from the calculated 63 kDa due to post-translational modifications .

What controls should be included when using IL10RA antibodies?

Proper controls are essential for antibody validation and experimental reliability:

• Positive controls: Include samples known to express IL10RA (e.g., HeLa cells, human heart tissue, human liver tissue)

• Negative controls: Include samples with low/no IL10RA expression or IL10RA knockout samples

• Loading controls: Use housekeeping proteins (e.g., β-actin, GAPDH) to normalize protein loading

• Isotype controls: Include appropriate isotype-matched control antibody to assess non-specific binding

• Blocking controls: Pre-incubate antibody with recombinant IL10RA protein to demonstrate specificity

• Secondary-only controls: Omit primary antibody to assess background from secondary antibody

For immunofluorescence studies specifically, pre-blocking with recombinant human IL-10 or unlabeled anti-IL10RA antibody before staining can demonstrate specificity .

How can IL10RA antibodies be used to study differential expression in inflammatory disorders?

IL10RA antibodies are valuable tools for investigating the dysregulation of anti-inflammatory pathways in various diseases:

• Expression analysis: Quantify IL10RA levels in affected tissues versus healthy controls using WB or IHC

• Cell-specific expression: Use flow cytometry with cell-type-specific markers to identify which immune cell populations show altered IL10RA expression

• Signaling pathway analysis: Combine with phospho-STAT3 detection to assess downstream signaling functionality

• Receptor regulation: Study how IL10RA expression changes during disease progression

• Therapeutic response markers: Monitor IL10RA expression as a biomarker for treatment efficacy

This is particularly relevant for inflammatory bowel diseases, where IL10R signaling plays a critical role in intestinal homeostasis. Studies have shown that defective IL10 signaling in antigen-presenting cells can result in proinflammatory states and loss of tolerance .

What approaches can be used to study the dynamics of IL10RA expression during immune cell activation?

Multiple methodologies can track changes in IL10RA expression during immune activation:

• Time-course analysis: Collect cells at different time points after activation stimuli (e.g., LPS, anti-CD3)

• Flow cytometry: Quantify surface IL10RA expression changes at single-cell resolution

• RT-qPCR: Monitor IL10RA mRNA expression changes relative to baseline

• Western blot: Assess total protein levels at different activation states

• Immunofluorescence microscopy: Visualize receptor redistribution and internalization

• Reporter systems: Generate IL10RA promoter-reporter constructs to study transcriptional regulation

This approach is valuable because IL10RA expression is dynamically regulated during immune responses. For example, naïve CD4+ T cells have low IL10RA expression that increases after activation, while human DCs downregulate IL10R1 after maturation .

Why might there be discrepancies between observed and predicted molecular weights of IL10RA?

The calculated molecular weight of IL10RA is 63 kDa (578 amino acids), but it typically appears at 70-72 kDa and 90-95 kDa in Western blots . These discrepancies can be attributed to:

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications can increase apparent molecular weight

• Protein isoforms: Alternative splicing may generate different protein variants

• Incomplete denaturation: Residual tertiary structure can affect protein migration

• Protein-protein interactions: Strong interactions that persist during SDS-PAGE

• Technical factors: Gel percentage, running conditions, and buffer composition can affect migration patterns

Researchers should validate antibody specificity using additional techniques such as immunoprecipitation followed by mass spectrometry or using genetic knockout controls.

How can researchers address non-specific binding issues with IL10RA antibodies?

Non-specific binding can compromise experimental results. To mitigate this:

• Optimize blocking conditions: Test different blocking agents (BSA, non-fat milk, commercial blockers) and concentrations

• Adjust antibody concentration: Titrate to find optimal dilution that maximizes specific signal while minimizing background

• Increase washing stringency: Use additional wash steps or higher detergent concentration

• Pre-adsorb antibody: Incubate with lysates from cells not expressing IL10RA to remove cross-reactive antibodies

• Use alternative detection methods: Switch from colorimetric to chemiluminescent or fluorescent detection for improved signal-to-noise ratio

• Include specificity controls: Use blocking peptides, isotype controls, or genetic knockouts to confirm specificity

For example, with the JES3-19F1 antibody, pre-blocking with recombinant human IL-10 or unlabelled JES3-19F1 antibody prior to staining can demonstrate specificity .

How are IL10RA antibodies being used to understand immune cell differentiation?

IL10RA antibodies are invaluable for studying how IL-10 signaling affects immune cell fate decisions:

• Macrophage polarization: IL10R signaling orchestrates the development of monocytes into different subsets of macrophages. During peritonitis, IL10 expression increases in early phases, directing monocytes to develop into MHCII low macrophages, while decreased IL10 in later phases promotes MHCII high macrophage differentiation

• T cell subset development: IL10RA signaling affects the balance between effector and regulatory T cell populations

• Dendritic cell maturation: IL10R1 expression is downregulated during DC maturation, enabling them to produce higher levels of proinflammatory mediators and prime T cells effectively

• Neutrophil activation states: While non-inflammatory neutrophils lack IL10Rα expression, stimulation with LPS or IL4 induces IL10Rα synthesis, allowing IL10 to modulate cytokine production

These applications highlight how IL10RA antibodies help track receptor expression as a marker for cellular differentiation states and functional outcomes.

What is the significance of IL10RA signaling in intestinal homeostasis?

IL10RA signaling is essential for maintaining intestinal immune balance:

• Epithelial integrity: IL10RA is constitutively expressed in colonic epithelial cells, suggesting a direct role in epithelial function and barrier maintenance

• Myeloid cell regulation: Intestinal macrophages constitutively produce IL10 and are generally hypo-responsive to TLR-mediated stimulation, contributing to mucosal homeostasis

• T cell tolerance: IL10-secreting CX3CR1highCD11b+CD11c+ cells suppress intestinal inflammation by inhibiting T-cell responses through IL10/STAT3-dependent mechanisms

• Disease relevance: Dysregulated IL10RA signaling is implicated in inflammatory bowel diseases, with studies showing that defective IL10 signaling in antigen-presenting cells results in proinflammatory states and loss of tolerance

IL10RA antibodies are critical tools for studying these processes in both normal physiology and disease states.