OSMR Antibody, Biotin conjugated

What is OSMR and what biological role does it play in cellular signaling?

OSMR (Oncostatin M-specific receptor subunit beta) is a critical transmembrane protein that functions as part of multiple cytokine receptor complexes. It associates with gp130 to form the type II OSM receptor that specifically responds to Oncostatin M. Additionally, OSMR associates with gp130-like receptor (GPL) to form a receptor complex that responds to IL-31 . The human OSMR beta protein has a molecular weight of 150-180 kDa and belongs to the IL-6 receptor family .

At the molecular level, OSMR beta contains multiple structural domains including one partial and one complete hematopoietin domain, an Ig-like domain, and three fibronectin type-III domains. The cytoplasmic portion contains signaling motifs known as box1, 2, and 3 . When activated, OSMR initiates signaling through multiple pathways, particularly the Jak/STAT and MAPK cascades. Notably, OSMR beta-containing receptors can activate STAT5b and SHC, which distinguishes them from other IL-6 family receptors .

What is the structural composition of OSMR Antibody, Biotin conjugated?

OSMR Antibody, Biotin conjugated is a rabbit polyclonal antibody specifically targeting the human Oncostatin M-specific receptor subunit beta (OSMR) that has been chemically linked to biotin molecules . The antibody is developed using recombinant Human Oncostatin-M-specific receptor subunit beta protein (specifically amino acids 120-193) as the immunogen .

The immunoglobulin isotype is IgG, and it's provided in liquid form with a diluent buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4 . Some versions of this antibody undergo protein G purification with >95% purity . The biotin conjugation enables versatile detection methods through the strong biotin-streptavidin interaction system, allowing researchers to pair this antibody with various streptavidin-linked detection reagents.

How does OSMR Antibody differ from anti-Oncostatin M antibody?

While these antibodies target related molecules within the same signaling pathway, they recognize different proteins:

• OSMR Antibody targets the Oncostatin M receptor (specifically the beta subunit), which is a transmembrane receptor protein expressed on the surface of responsive cells. This antibody recognizes the receptor that binds to and responds to the cytokine Oncostatin M .

• Anti-Oncostatin M antibody targets Oncostatin M itself, which is the soluble cytokine ligand that binds to the receptor. Oncostatin M is a secreted protein that functions as a growth regulator, inhibiting proliferation of certain tumor cell lines while stimulating proliferation in others (such as AIDS-KS cells) .

This fundamental difference means these antibodies serve different research purposes - OSMR antibodies are used to study receptor expression and distribution, while anti-Oncostatin M antibodies examine the cytokine's presence and concentration.

What are the validated applications for OSMR Antibody, Biotin conjugated?

According to the product information, OSMR Antibody, Biotin conjugated has been validated primarily for Enzyme-Linked Immunosorbent Assay (ELISA) applications . While this represents the manufacturer-validated application, researchers should note that biotin-conjugated antibodies are generally versatile tools that can potentially be utilized in other detection systems.

Some related OSMR beta antibodies with different conjugates (such as APC-conjugated antibodies) have been validated for flow cytometry applications, as demonstrated by their successful use in detecting OSMR beta in HeLa human cervical epithelial carcinoma cell lines . This suggests that with proper optimization, biotin-conjugated variants might also be applicable to flow cytometry using streptavidin-fluorophore detection systems.

For any application beyond the validated ELISA, researchers should conduct thorough validation studies with appropriate positive and negative controls to confirm specificity and performance.

What is the recommended protocol for using OSMR Antibody, Biotin conjugated in ELISA?

While specific manufacturer protocols should be consulted, a general methodological approach for using biotin-conjugated OSMR antibody in ELISA includes:

• Coating: Coat microplate wells with capture antibody (if using a sandwich ELISA) or antigen (if detecting OSMR directly).

• Blocking: Block remaining protein-binding sites with a suitable blocking buffer (typically BSA-based or casein-based) to prevent non-specific binding.

• Sample addition: Add samples containing the target protein.

• Primary antibody incubation: Add the biotin-conjugated OSMR antibody at an optimized dilution (titration experiments are recommended to determine this).

• Detection reagent: Add streptavidin-HRP (horseradish peroxidase) which binds with high affinity to the biotin conjugate.

• Substrate addition: Add appropriate substrate for HRP (such as TMB).

• Signal measurement: Measure the colorimetric signal using a spectrophotometer.

Between each step, thorough washing with PBS-T (PBS containing 0.05-0.1% Tween-20) is essential to remove unbound reagents. As noted in the product information, optimal dilutions should be determined by each laboratory for their specific experimental conditions .

How can OSMR Antibody be used in flow cytometry applications?

Although the biotin-conjugated OSMR antibody in the search results is primarily validated for ELISA, related OSMR beta antibodies have been successfully used in flow cytometry. The methodological approach for using OSMR antibodies in flow cytometry typically involves:

• Cell preparation: Harvest cells of interest (e.g., HeLa cells as demonstrated in the search results ) and adjust to 1 × 10^6 cells/mL in flow cytometry staining buffer.

• Blocking: Incubate cells with Fc blocking reagent to reduce non-specific binding.

• Primary antibody staining: If using the biotin-conjugated antibody, add at an optimized concentration and incubate (typically 30-60 minutes at 4°C).

• Secondary detection: Add fluorophore-conjugated streptavidin (e.g., streptavidin-PE or streptavidin-APC) and incubate according to manufacturer's recommendations.

• Washing: Wash cells thoroughly between steps to remove unbound antibody.

• Analysis: Analyze by flow cytometry alongside appropriate controls.

The search results demonstrate successful detection of OSMR beta in HeLa cells using an APC-conjugated antibody, which produced a clear shift in fluorescence intensity compared to the isotype control . For validation purposes, a similar experiment using OSMR beta knockout HeLa cells showed no staining, confirming antibody specificity .

How should I validate the specificity of OSMR Antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable research data. For OSMR Antibody, a comprehensive validation approach should include:

• Positive and negative control samples: Use cell lines or tissues known to express or lack OSMR. HeLa cells have been demonstrated as a positive control for OSMR beta expression .

• Genetic knockout validation: The search results describe using OSMR beta knockout HeLa cells as a negative control, which showed no staining with the antibody . This represents the gold standard for specificity validation.

• Signal peptide blocking: Pre-incubate the antibody with excess immunogen peptide before application to samples. Specific binding should be reduced or eliminated.

• Western blot analysis: If performing Western blot, the antibody should detect a band at the expected molecular weight (150-180 kDa for OSMR beta ).

• Cross-reactivity assessment: Test the antibody on samples from different species if working with non-human models. The provided antibody is specified for human reactivity .

• Isotype control comparison: Include an isotype-matched, non-specific antibody with the same conjugate to identify background or non-specific staining levels.

These validation steps should be documented and included in research methods to demonstrate antibody reliability.

What are the optimal storage conditions for maintaining OSMR Antibody, Biotin conjugated activity?

To preserve antibody activity and prevent degradation, OSMR Antibody, Biotin conjugated should be stored according to these guidelines:

• Temperature: Store at -20°C or -80°C for long-term storage .

• Aliquoting: Upon receipt, divide the antibody into small working aliquots to avoid repeated freeze-thaw cycles .

• Light protection: Keep biotin-conjugated antibodies protected from prolonged exposure to light, as biotin conjugates can be photosensitive .

• Preparation before use: Gently mix the antibody solution before use. Spinning the vial briefly before opening is recommended to collect liquid that may have accumulated on the cap or sides .

• Avoid repeated freeze-thaw cycles: Each freeze-thaw cycle can reduce antibody activity. The search results specifically caution against repeated freezing and thawing .

• Buffer considerations: The antibody is provided in a buffer containing 50% glycerol, which helps maintain stability during freeze-thaw transitions .

Following these storage recommendations will help ensure consistent antibody performance across experiments and maximize the usable lifespan of the reagent.

How do I determine the optimal working concentration for my specific experiment?

Determining the optimal working concentration of OSMR Antibody, Biotin conjugated requires systematic titration experiments tailored to your specific application. A methodological approach includes:

• Initial range selection: Based on manufacturer recommendations, select a range of concentrations to test. For antibodies, typically start with a 2-fold or 5-fold dilution series.

• Application-specific titration:

  • For ELISA: Prepare a standard curve using recombinant OSMR protein at known concentrations. Test each antibody dilution against this curve to determine which concentration provides the best combination of sensitivity (low background) and dynamic range.

  • For flow cytometry: Test multiple antibody concentrations on positive control cells (e.g., HeLa cells ) and plot the signal-to-noise ratio for each concentration.

• Negative control inclusion: For each concentration tested, include appropriate negative controls (isotype controls and/or cells known not to express OSMR) to assess non-specific binding.

• Signal-to-noise calculation: Calculate the ratio between specific signal and background for each concentration.

• Saturation analysis: Plot a binding curve to identify the saturation point. The optimal concentration is typically just before saturation, providing strong specific signal while economizing antibody usage.

• Reproducibility verification: Once an optimal concentration is identified, verify its reproducibility across multiple experiments.

The search results emphasize that "optimal dilutions or concentrations should be determined by the scientist" , recognizing that experimental conditions vary between laboratories and applications.

How do polyclonal and monoclonal OSMR antibodies compare in research applications?

The choice between polyclonal and monoclonal OSMR antibodies significantly impacts experimental outcomes and should be based on specific research requirements:

Polyclonal OSMR Antibodies (like the biotin-conjugated version in the search results ):

• Recognize multiple epitopes on the OSMR protein, potentially increasing detection sensitivity

• May provide more robust detection when protein conformation is altered or partially denatured

• Batch-to-batch variation can occur due to the heterogeneous nature of the antibody population

• Ideal for applications where maximum signal amplification is desired, such as immunohistochemistry or detecting low-abundance targets

Monoclonal OSMR Antibodies (like the research grade vixarelimab biosimilar mentioned ):

• Recognize a single epitope with high specificity

• Provide consistent performance with minimal batch-to-batch variation

• May be less sensitive than polyclonals for certain applications

• Superior for quantitative applications where reproducibility is critical

• Preferred for applications requiring discrimination between highly similar proteins or epitopes

When designing experiments, researchers should consider that polyclonal antibodies like the biotin-conjugated OSMR antibody described in the search results may provide enhanced signal detection due to their multi-epitope binding capability, while monoclonal alternatives offer greater specificity and reproducibility.

What strategies can I employ to reduce background when using biotin-conjugated antibodies?

Biotin-conjugated antibodies can sometimes generate background signals, particularly in tissues or cells that contain endogenous biotin. To minimize background and optimize signal-to-noise ratio:

• Block endogenous biotin: Pretreat samples with streptavidin followed by free biotin to block endogenous biotin sites before adding the biotin-conjugated primary antibody.

• Optimize antibody concentration: Use the minimum concentration of biotin-conjugated antibody that yields satisfactory specific signal, as determined through titration experiments.

• Include appropriate blocking buffers: Use protein-based blocking buffers (containing BSA, casein, or serum) to reduce non-specific binding sites before antibody application.

• Adjust washing conditions: Implement more stringent washing steps (increased number of washes, higher salt concentration, or addition of detergents like Tween-20) to remove unbound antibody.

• Use avidin/streptavidin conjugates with low background: Some streptavidin preparations have lower non-specific binding properties than others.

• Consider alternative detection methods: If background remains problematic despite optimization, consider using directly conjugated primary antibodies or alternative detection systems.

• Include proper controls: Always run parallel samples with isotype control antibodies to assess the level of non-specific binding.

These strategies should be systematically tested and optimized for each specific experimental system and application.

How can I investigate the functional relationship between OSMR and IL-31 signaling pathways?

The search results indicate that OSMR associates with IL31RA to form the IL31 receptor, highlighting an important functional relationship . To investigate this relationship, researchers can implement several methodological approaches:

• Co-immunoprecipitation studies: Use antibodies against OSMR to pull down protein complexes, then probe for IL-31RA to confirm physical interaction. Alternatively, perform the reciprocal experiment using IL-31RA antibodies.

• Proximity ligation assays: Employ this technique to visualize and quantify protein-protein interactions between OSMR and IL-31RA in situ at the single-molecule level.

• FRET/BRET analysis: Use fluorescence or bioluminescence resonance energy transfer to examine the proximity of labeled OSMR and IL-31RA proteins in living cells.

• Stimulation experiments: Treat cells with IL-31 and assess STAT3 activation (and possibly STAT1 and STAT5 ), as OSMR signals through these pathways.

• Receptor knockdown/knockout studies: Use siRNA, CRISPR-Cas9, or other genetic tools to selectively reduce or eliminate OSMR expression, then examine effects on IL-31 signaling responses.

• Comparative signaling analysis: Compare signaling patterns when cells are stimulated with Oncostatin M versus IL-31 to identify shared and distinct signaling events.

• Receptor chimeras: Create chimeric receptors with domains from OSMR and other related receptors to identify regions critical for IL-31 signaling specificity.

• Biotin-conjugated antibody applications: The biotin-conjugated OSMR antibody could be useful for isolating receptor complexes through streptavidin-based pulldown methods to study receptor complex composition under different stimulation conditions.

This multifaceted approach provides complementary data on both the physical interactions and functional consequences of OSMR-IL31RA associations.

What factors should I consider when designing multicolor flow cytometry panels that include OSMR detection?

When incorporating OSMR detection into multicolor flow cytometry panels, several technical considerations are essential for generating reliable data:

• Fluorophore selection and spectral overlap: If using biotin-conjugated OSMR antibody, select a streptavidin-fluorophore conjugate that minimizes spectral overlap with other fluorophores in your panel. Consider brightness requirements based on expected OSMR expression levels.

• Panel design hierarchy: Position OSMR in your panel based on its importance to your research question and expected expression level. Critical markers with low expression may require brighter fluorophores.

• Controls for compensation: Include single-stained controls for each fluorophore in your panel, including the fluorophore used for OSMR detection, to enable accurate compensation.

• FMO controls: Prepare Fluorescence Minus One controls to properly set gates, especially important for markers with continuous expression patterns like many receptors.

• Titration for each antibody: Determine optimal concentration for every antibody in the panel within the context of the full staining protocol, as antibodies may perform differently in complex mixtures.

• Blocking strategy: Include appropriate Fc receptor blocking to prevent non-specific binding, particularly important when working with primary cells.

• Sample preparation considerations: Optimize fixation and permeabilization protocols if detecting both surface OSMR and intracellular markers.

• Viability discrimination: Include a viability dye to exclude dead cells, which can bind antibodies non-specifically.

• Antibody order: When using biotin-streptavidin systems alongside directly conjugated antibodies, the staining sequence may affect results. Test different staining sequences to optimize signal.

The search results show successful detection of OSMR beta in HeLa cells using APC-conjugated antibodies, with clear discrimination between positive and negative populations , demonstrating that with proper panel design, OSMR can be effectively incorporated into flow cytometry experiments.

How might OSMR detection contribute to understanding pathological conditions and potential therapeutic applications?

OSMR detection using specific antibodies can provide valuable insights into various pathological conditions where this receptor plays significant roles:

• Cancer research: The search results indicate that OSM (Oncostatin M) inhibits proliferation in certain tumor cell lines while stimulating it in others (like AIDS-KS cells) . Detecting OSMR expression patterns across cancer types could help predict responsiveness to OSM-based therapies or identify new therapeutic targets.

• Inflammatory diseases: OSMR mediates signaling through the Jak/STAT and MAPK pathways , which are central to many inflammatory processes. Quantifying OSMR expression in inflammatory conditions could help stratify patients for targeted therapies.

• Hematological disorders: The search results mention that loss of OSMR beta expression in mice blocks erythroid progenitor development and reduces circulating platelets and erythrocytes , suggesting its importance in hematological development. OSMR detection could provide insights into certain blood disorders.

• Bone metabolism: The type II OSM receptor (which includes OSMR) is noted as the only IL-6 family receptor that promotes osteoblast differentiation , indicating a potential role in bone disorders.

• IL-31-related pathologies: Given OSMR's role in forming the IL-31 receptor complex , detecting its expression could be relevant for conditions where IL-31 signaling is implicated, such as atopic dermatitis and pruritus.

• Therapeutic antibody development: The mention of vixarelimab (an OSMR beta antibody) suggests ongoing development of therapeutic antibodies targeting this receptor. Research-grade antibodies like those described in the search results are essential tools for developing and validating such therapeutics.

• Biomarker development: Changes in OSMR expression or localization could potentially serve as biomarkers for disease progression or treatment response.

Utilizing biotin-conjugated OSMR antibodies enables flexible detection methods across multiple experimental platforms, facilitating comprehensive studies of OSMR biology in both basic research and translational medicine contexts.