IL1RL1 Antibody

What is IL1RL1 and what is its molecular structure?

IL1RL1 (also known as ST2, IL-33R, and T1) is a member of the Toll-like receptor superfamily and functions as the receptor for Interleukin-33. It is a 556 amino acid single-pass type I membrane protein with a molecular weight of approximately 63.4 kDa . The protein undergoes post-translational modifications, notably glycosylation, which can affect its observed molecular weight in experimental conditions. In Western blot analyses, IL1RL1 may appear at different molecular weights ranging from 50-63 kDa depending on the extent of glycosylation and the specific isoform being detected .

What are the different forms of IL1RL1 and how do they function?

IL1RL1 exists in two main forms as splice variants:

The membrane-bound form (ST2L) signals through the MyD88/NF-κB pathway upon binding IL-33, while the soluble form (sST2) acts as a decoy receptor that prevents IL-33 from binding to the membrane-bound receptor, effectively inhibiting IL-33 signaling . The signaling through ST2L requires association with the co-receptor IL1RAP and recruits MYD88, IRAK1, IRAK4, and TRAF6 .

Which cell types express IL1RL1?

IL1RL1 is expressed by multiple immune and non-immune cell types:

• Immune cells: Th2 lymphocytes, mast cells, basophils, eosinophils, invariant NKT cells, NK cells, and regulatory T cells

• Non-immune cells: Smooth muscle cells, endothelial cells, epithelial cells, and cardiomyocytes

The expression of IL1RL1 on these various cell types contributes to its diverse roles in inflammation, allergy, and tissue homeostasis .

What are the optimal applications for detecting IL1RL1 with antibodies?

Based on the search results, IL1RL1 antibodies are successfully used in multiple applications with specific dilution recommendations:

For immunohistochemistry, researchers should note that antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may also be used as an alternative .

How should I validate the specificity of my IL1RL1 antibody?

Proper validation of IL1RL1 antibodies is critical for research reproducibility:

• Genetic knockdown/knockout controls: Use IL1RL1 knockout or knockdown samples as negative controls . Search results mention published applications using KD/KO approaches for antibody validation.

• Multiple detection methods: Confirm findings using at least two different techniques (e.g., WB plus IHC or flow cytometry).

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate specificity.

• Positive control tissues: Use tissues known to express IL1RL1, such as placenta, lung tissue, or heart tissue as demonstrated in the search results .

• Molecular weight verification: Confirm that detected bands correspond to expected molecular weights (63 kDa for full-length IL1RL1, with variants at 37 kDa and 30 kDa) .

How can I distinguish between soluble and membrane-bound forms of IL1RL1?

Distinguishing between sST2 and ST2L requires specific methodological approaches:

• Western blot analysis: The two forms have different molecular weights. Use gel conditions that can resolve these differences, typically 8-10% SDS-PAGE gels run at lower voltage for better separation .

• Cell fractionation: Isolate membrane fractions to enrich for ST2L before antibody detection.

• ELISA techniques: For sST2, use ELISA kits specifically designed to detect the soluble form in serum or other body fluids. The R&D Quantikine® ELISA Human ST2/IL-1-R4 kit has been validated for this purpose .

• Flow cytometry: For detecting membrane-bound ST2L on cell surfaces, use non-permeabilized cells and antibodies targeting the extracellular domain .

• Immunofluorescence microscopy: Membrane-bound ST2L will show distinct membrane localization, while diffuse cytoplasmic staining may indicate the presence of the soluble form prior to secretion.

What is the relationship between IL1RL1 genetic polymorphisms and disease?

IL1RL1 genetic polymorphisms have been associated with various allergic conditions:

These genetic variations can affect antibody binding and experimental outcomes. Researchers studying IL1RL1 in disease contexts should consider genotyping their samples or using cell lines with known IL1RL1 genotypes to account for potential variability .

How can IL1RL1 antibodies help investigate the role of this receptor in leukemia?

IL1RL1 has been identified as dynamically expressed on leukemia stem cells, particularly in AML with the CBFB-MYH11 fusion oncogene:

• Flow cytometry sorting: Use fluorescently-labeled IL1RL1 antibodies to isolate IL1RL1+ and IL1RL1- leukemia cell populations for functional assays .

• Survival assays: Research has shown that IL1RL1+ cells from CBFB-MYH11+ leukemia demonstrate enhanced survival following chemotherapy compared to IL1RL1- cells .

• IL-33 stimulation studies: Treat sorted IL1RL1+ cells with IL-33 to investigate how IL-33/ST2 signaling affects leukemia cell proliferation, survival, and expression of pro-survival proteins .

• Serial transplantation experiments: Use IL1RL1 antibodies to track the dynamics of IL1RL1 expression in leukemia stem cell populations through multiple passages in animal models .

Research findings indicate that IL1RL1 is not restricted to a specific stage of differentiation in leukemia cells but is dynamically expressed and promotes cell survival, suggesting it as a potential therapeutic target .

What are the optimal sample preparation methods for Western blot detection of IL1RL1?

For optimal Western blot detection of IL1RL1:

• Lysis buffer selection: Use RIPA or NP-40 based buffers containing protease inhibitors to prevent degradation.

• Protein denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer containing SDS and β-mercaptoethanol to ensure proper denaturation.

• Gel percentage: Use 8-10% SDS-PAGE gels for better resolution of the 63 kDa IL1RL1 protein.

• Transfer conditions: Transfer to PVDF membranes at lower voltage (30V) overnight at 4°C to ensure complete transfer of higher molecular weight proteins.

• Blocking: Block membranes in 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature to reduce background signal.

• Antibody dilution: For polyclonal antibodies, use 1:500-1:2000 dilution; for monoclonal antibodies, a wider range of 1:5000-1:50000 can be used depending on the specific antibody .

• Detection system: Use a sensitive chemiluminescent detection system compatible with your antibody's host species.

How can I optimize IL1RL1 detection in immunohistochemistry/immunofluorescence?

For optimal IHC/IF detection of IL1RL1:

• Fixation: Use 4% paraformaldehyde for tissue fixation to preserve antigen integrity.

• Antigen retrieval: Evidence suggests that TE buffer at pH 9.0 works best for IL1RL1 detection, though citrate buffer at pH 6.0 can be used as an alternative .

• Blocking: Block with 5-10% normal serum from the same species as the secondary antibody for 1 hour to reduce non-specific binding.

• Antibody dilution: Use 1:1000-1:4000 dilution for IHC applications .

• Incubation conditions: Incubate primary antibody overnight at 4°C to maximize specific binding while minimizing background.

• Controls: Include positive control tissues (heart, lung, or placenta) and negative controls (secondary antibody only) in each experiment .

• Counterstaining: Use hematoxylin for nuclear visualization in IHC or DAPI for nuclear visualization in IF.

How can I troubleshoot non-specific binding issues with IL1RL1 antibodies?

When encountering non-specific binding with IL1RL1 antibodies:

• Increase antibody dilution: Try more dilute antibody concentrations to reduce non-specific binding.

• Optimize blocking: Increase blocking time or concentration, or try different blocking agents (BSA, normal serum, commercial blocking solutions).

• Increase washing steps: Add additional washing steps with TBST or PBS-T to remove unbound antibody.

• Pre-absorb antibody: Incubate antibody with non-relevant tissue lysate to remove antibodies that bind non-specifically.

• Use monoclonal antibodies: Consider switching to monoclonal antibodies for higher specificity .

• Verify using genetic controls: Confirm specificity using IL1RL1 knockout or knockdown samples as true negative controls .

• Change detection system: Try different secondary antibodies or detection systems that may offer improved signal-to-noise ratios.

How is IL1RL1 involved in allergic conditions?

IL1RL1 plays significant roles in allergic conditions through several mechanisms:

• TH2 inflammation: IL1RL1 signaling drives cytokine production in Th2 lymphocytes and type 2 innate immune cells, which are critical for allergic responses .

• Mast cell activation: After IgE-crosslinking, mast cell activation, migration, and degranulation are significantly enhanced by stimulation of the membrane-bound IL1RL1 (ST2L) .

• Eosinophilic inflammation: IL-1RL1a serum levels predict the development of eosinophilic asthma characterized by high FeNO (fractional exhaled nitric oxide) in preschool wheezing children .

• Food allergy pathogenesis: IL-33 has been shown to be critical for the development of gastrointestinal food allergy in a mouse model, acting through IL1RL1 .

Research has shown that IL1RL1 genetic polymorphisms are associated with specific food allergies, including peanut allergy (rs1041973) and chicken egg sensitization (rs1420101) .

How can IL1RL1 antibodies be used to study neutrophilic inflammation?

While IL1RL1 is primarily associated with type 2 inflammation, recent research has revealed its role in neutrophilic inflammation:

• Bacterial infection models: IL1RL1 promotes airway bacterial clearance by enhancing neutrophil recruitment. Using IL1RL1 antibodies, researchers can differentiate IL1RL1+ and IL1RL1- populations to study their differential responses to bacterial challenges like Mycoplasma pneumoniae .

• Viral infection models: In human rhinovirus (HRV) infection models, IL1RL1 knockout mice showed altered levels of neutrophil-recruiting chemokines like KC and IP-10, suggesting IL1RL1's influence on neutrophilic responses to viral infection .

• Chemokine analysis: Use IL1RL1 antibodies in combination with chemokine assays to correlate IL1RL1 expression with neutrophil-attracting chemokine production in various cell types.

• Time-course experiments: IL1RL1 antibodies can be used to track the dynamics of IL1RL1 expression during infection progression, as the research indicates differential effects at 16 hours versus 72 hours post-infection .

Methodologically, researchers can use IL1RL1 antibodies to isolate and characterize cell populations with differential IL1RL1 expression, then assess their production of neutrophil-recruiting factors and response to pathogenic stimuli .