OSMR Antibody, HRP conjugated

What is OSMR and what biological functions does it mediate?

OSMR (Oncostatin M-specific receptor subunit beta) is a single-pass membrane protein belonging to the type I cytokine receptor family. It functions as a component of two distinct receptor complexes: it partners with gp130 to form the OSM receptor complex, and it associates with IL31RA to form the IL31 receptor. Through these complexes, OSMR mediates signaling via several pathways, most notably activating STAT3, and potentially STAT1 and STAT5 . OSMR mRNA is expressed at relatively high levels in neural cells, fibroblasts, epithelial cells, and various tumor cell lines . Functionally, OSMR activation is involved in cellular proliferation, differentiation, and has emerging roles in cancer progression.

What applications are OSMR antibodies suitable for?

Based on validated research data, OSMR antibodies have demonstrated utility in multiple experimental applications:

• Western Blot (WB): Effective dilution ranges from 1:1000-1:4000

• Immunohistochemistry (IHC): Validated in multiple publications

• Immunofluorescence (IF): Confirmed in at least 4 published studies

• Flow Cytometry: Successfully used for detecting OSMR in cell lines such as HeLa

• ELISA: Particularly as detection antibodies when paired with appropriate capture antibodies

• Cross-linking dimerization assays: For investigating receptor complex formation

How can I validate the specificity of an OSMR antibody?

The gold standard for antibody validation involves using knockout systems. According to search results, researchers have successfully validated OSMR antibody specificity using:

• Knockout cell line comparison: Western blot analysis comparing parental HeLa cells with OSMR knockout HeLa cells shows specific detection of OSMR at the expected molecular weight in parental cells but absence of signal in knockout cells .

• Flow cytometry validation: Flow cytometry comparing parental and OSMR knockout HeLa cells demonstrates specific staining in parental cells but absence of staining in knockout cells .

• Isotype control comparison: Using appropriate isotype control antibodies (e.g., MAB002) in parallel with OSMR-specific antibodies to confirm specific staining patterns .

What is the typical molecular weight observed for OSMR in Western blotting?

• 110 kDa (reported by Proteintech for their 10982-1-AP antibody)

• 45 kDa (reported for a specific band detected by MAB4389)

• 150-180 kDa (reported by Abcam for their EPR24611-71 antibody)

These variations may reflect differences in post-translational modifications, splice variants, proteolytic processing, or detection of different domains of the protein. The expression pattern and observed molecular weight are consistent with published literature (PMID: 8999038) .

What are the key advantages of HRP-conjugated OSMR antibodies?

HRP-conjugated OSMR antibodies offer several methodological advantages:

• Direct detection without secondary antibodies, reducing experimental time and potential background issues

• Improved signal-to-noise ratio in many detection systems

• Compatible with various substrates (chemiluminescent, colorimetric, or fluorescent)

• Reduced cross-reactivity issues common with two-antibody detection systems

• Greater consistency in quantitative applications due to fixed enzyme-to-antibody ratio

When using HRP-conjugated antibodies, researchers should optimize dilutions for each experimental system to obtain optimal results .

How do OSMR antibodies perform in detecting OSMR-mediated receptor complex formation?

OSMR forms heterodimeric complexes with other receptor subunits, and specialized approaches are needed to study these interactions. Research data shows:

• Cross-linking dimerization assay: Treatment with OSM followed by a membrane-impermeable chemical crosslinker (BS3) allows for detection of OSMR-IL6ST (gp130) heterodimeric receptor complexes. In cisplatin-resistant ovarian cancer cells (A2780-CisR), OSM-induced heterodimerization of OSMR was relatively higher than in cisplatin-sensitive A2780 cells, correlating with increased OSMR and OSM expression in resistant cells .

• Co-immunoprecipitation: OSMR antibodies successfully capture OSMR-IL6ST dimerized receptor complexes along with monomeric OSMR when cells are treated with OSM, enabling investigation of receptor complex formation dynamics .

• Detection of IL31 receptor complex: OSMR associates with IL31RA to form the heterodimeric IL31 receptor. Appropriate antibodies can be used to study this complex formation and subsequent activation of STAT3 and possibly STAT1 and STAT5 .

What approaches work best for studying OSMR's role in cancer resistance mechanisms?

OSMR has emerged as a promising target in cancer research, particularly in cisplatin resistance in ovarian cancer. Research strategies include:

• Correlation analysis: In TCGA ovarian cancer datasets, OSMR shows high positive correlation with integrin genes (ITGAV, ITGA3, ITGA5, ITGB1, ITGB3, ITGB4, ITGB5, and ITGB8), suggesting a mechanistic relationship .

• Ectopic expression and knockdown studies: These approaches have demonstrated that OSMR directly regulates integrin gene expression (ITGAV and ITGB3) through STAT3 activation .

• Therapeutic targeting: Anti-OSMR human antibody treatment inhibits growth and metastasis of ovarian cancer cells and sensitizes them to cisplatin treatment, indicating a potential therapeutic approach .

• Comparing resistant vs. sensitive cell lines: Comparing OSMR expression and signaling between cisplatin-resistant and cisplatin-sensitive cell lines reveals mechanistic insights into resistance development .

How can species-specific activation of OSMR be studied using antibodies?

The species specificity of OSMR activation is an important consideration for translational research. Studies show:

• AB loop mutations: The AB loop of oncostatin M (OSM) determines species-specific receptor activation. Chimeric proteins with mouse-human AB loop replacements can activate both human OSMR and human LIFR .

• Critical residue identification: Key amino acid residues (such as Gly-39 in human OSM) are critical for human OSMR activation, while human LIFR activation has more relaxed ligand requirements .

• Receptor activation assays: STAT3 phosphorylation, TIMP1 expression, and cell proliferation inhibition assays can be used to assess receptor activation by different OSM variants .

When using antibodies to study these interactions, researchers should select antibodies that recognize the appropriate species-specific domains and epitopes.

What are the best sample preparation methods for OSMR detection in different applications?

Optimal sample preparation depends on the application:

• Western Blot:

  • Buffer recommendation: RIPA or NP-40 based lysis buffers with protease inhibitors

  • Reducing conditions are recommended (as shown in the scientific data for MAB4389)

  • Immunoblot Buffer Group 1 was successfully used in published protocols

  • Loading control recommendations include GAPDH (MAB5718)

• Flow Cytometry:

  • Blocking recommendations: Follow protocols for staining membrane-associated proteins

  • Secondary antibody options include Allophycocyanin-conjugated Anti-Mouse IgG F(ab')2 or APC-conjugated Goat anti-Mouse IgG

• ELISA:

  • Antibody pairing: MAB4389 functions as an ELISA detection antibody when paired with Mouse Anti-Human OSM R beta Monoclonal Antibody (MAB43891)

  • Blocking recommendation: 5% NFDM/TBST has been reported effective

How do I troubleshoot weak or non-specific signals when using OSMR antibodies?

When encountering detection issues:

• Antibody dilution optimization: Titrate antibodies in each testing system to obtain optimal results; recommended ranges for Western blot are 1:1000-1:4000

• Expression level awareness: Some tissues express OSMR at low levels (e.g., human tonsil), requiring longer exposure times (3 minutes reported for ab282577)

• Specific band identification: Be aware of the expected molecular weight range (may vary from 45 kDa to 180 kDa depending on the antibody and detection conditions)

• Blocking optimization: 5% NFDM/TBST has been successfully used, but optimization may be required for different sample types

• Knockout/knockdown controls: Include appropriate positive and negative controls, particularly OSMR knockout cell lines when available, to confirm specificity

How can I use OSMR antibodies to investigate STAT3 signaling pathways?

OSMR signaling prominently activates the STAT3 pathway, which can be studied through:

• Phospho-STAT3 detection: Following OSMR activation with OSM or other ligands, detect phosphorylated STAT3 using phospho-specific antibodies. The timing matters - studies show clear signals after 10-minute stimulation .

• STAT3-responsive gene expression: Monitor downstream gene targets of STAT3, such as TIMP1, which shows increased expression 24 hours after stimulation in OSMR-expressing cells .

• Integrin gene regulation: OSMR directly regulates integrin gene expression (ITGAV and ITGB3) through STAT3 activation, which can be monitored using appropriate antibodies after OSMR stimulation .

• Pathway inhibitor studies: Combine OSMR antibodies with specific inhibitors of STAT3 or other pathway components to dissect signaling mechanisms.

• Time-course experiments: Both short-term (minutes for phosphorylation events) and long-term (24 hours for gene expression changes) time points are informative for understanding OSMR-STAT3 signaling dynamics .

What controls are essential when using OSMR antibodies in functional studies?

Rigorous controls ensure reliable results:

• Isotype controls: For flow cytometry and immunostaining, appropriate isotype control antibodies (e.g., MAB002) should be used in parallel with OSMR-specific antibodies .

• Loading controls: For Western blotting, GAPDH (e.g., MAB5718) serves as an effective loading control .

• Genetic controls: OSMR knockout cell lines provide the gold standard for antibody specificity validation. Studies have used OSMR knockout HeLa cells to confirm specificity in Western blot and flow cytometry .

• Stimulation controls: Include both unstimulated and OSM-stimulated samples to confirm receptor activation and downstream signaling events .

• Cross-species controls: When studying species specificity, include appropriate controls from different species to confirm antibody cross-reactivity or specificity .

How can I use OSMR antibodies to study receptor dimerization and complex formation?

Receptor complex formation is critical to OSMR function:

• Cross-linking approach: Treat cells with OSM followed by a membrane-impermeable chemical crosslinker (BS3), then perform immunoprecipitation with OSMR antibodies to capture heterodimeric complexes .

• Co-immunoprecipitation: OSMR antibodies can capture both monomeric OSMR and OSMR-partner complexes (such as OSMR-IL6ST), particularly after ligand stimulation .

• Flow cytometry: Surface expression of receptor components can be monitored before and after ligand stimulation to track internalization or expression changes .

• Comparison across cell lines: Comparative analysis between different cell types (e.g., cisplatin-sensitive vs. resistant cancer cells) can reveal differences in complex formation efficiency related to functional outcomes .

What are the key considerations for studying OSMR in cancer models?

OSMR has emerging roles in cancer biology:

• Correlation with clinical outcomes: OSMR expression correlates with integrin expression patterns in datasets like TCGA ovarian cancer and GSE45553 OVCAR8-CisR spheroid datasets .

• Therapeutic targeting approach: Anti-OSMR human antibody treatment inhibits growth and metastasis of ovarian cancer cells and sensitizes them to cisplatin treatment .

• Resistance mechanism studies: OSMR fosters expression of specific integrin genes, creating a crosstalk between OSMR and integrins that contributes to cisplatin resistance in ovarian cancer .

• Cell line selection: Cell lines with documented OSMR expression include HeLa (cervical carcinoma), A375 (melanoma), HEK-293T, Raji, and HepG2 (hepatocellular carcinoma) .

• Functional assays: Cell proliferation inhibition assays can assess the functional impact of OSMR targeting or activation in cancer models .

What are recommended protocols for using OSMR antibodies in Western blotting?

Optimized Western blotting protocols based on published data:

For optimizing HRP-conjugated OSMR antibodies specifically, eliminate the secondary antibody step and adjust dilution according to the conjugate's specific activity.

How should I optimize OSMR antibody dilutions for different experimental applications?

Dilution optimization recommendations:

• Western Blot: Start with 1:1000-1:4000 dilution range and adjust based on signal strength and background. For HRP-conjugated antibodies, higher dilutions (1:2000-1:10000) may be appropriate depending on the conjugation efficiency .

• Flow Cytometry: Begin with manufacturer's recommendations, typically 1-10 μg/ml for unconjugated antibodies. For HRP-conjugated variants, consider starting at 1:100-1:500 .

• Immunofluorescence: Start with 1-10 μg/ml for unconjugated antibodies and adjust based on signal-to-noise ratio .

• ELISA: For detection antibodies, initial dilutions of 1:1000-1:5000 are typical, but optimization is essential for each assay platform .

• Immunoprecipitation: Higher concentrations are typically required (2-5 μg per sample) .

Remember that "optimal dilutions should be determined by each laboratory for each application" , and sample type can significantly impact optimal dilution requirements.

What is the recommended storage and handling of OSMR antibodies to maintain activity?

Based on manufacturer recommendations:

• Storage temperature: Store at -20°C for optimal stability. Most formulations remain stable for one year after shipment when stored properly .

• Aliquoting considerations: For larger volumes, aliquoting is recommended to avoid repeated freeze-thaw cycles, though some formulations specify that "aliquoting is unnecessary for -20°C storage" .

• Buffer composition: Many OSMR antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Some smaller sizes (20μl) may contain 0.1% BSA .

• Working solution handling: Keep on ice when in use and return to -20°C promptly after use.

• Conjugated antibody considerations: HRP-conjugated antibodies may have special storage requirements to maintain enzyme activity. Avoid repeated freeze-thaw cycles and exposure to light.

How can I use OSMR antibodies in multiplex detection systems?

For simultaneous detection of multiple targets:

• Antibody species selection: Choose primary antibodies raised in different host species to avoid cross-reactivity of secondary detection reagents.

• Fluorophore selection: For fluorescent multiplex systems, select fluorophores with minimal spectral overlap. If using HRP-conjugated OSMR antibodies, consider tyramide signal amplification (TSA) methods which allow sequential detection.

• Sequential detection protocol: For Western blots using HRP-conjugated antibodies, consider membrane stripping and reprobing, or using different visualization methods (chemiluminescence vs. colorimetric) for different targets.

• Optimization of antibody concentrations: Each antibody in the multiplex panel may require different concentrations to achieve balanced signal intensity.

• Controls for specificity: Include single-stain controls to confirm specificity and absence of cross-reactivity in multiplex systems.

What are the species cross-reactivity considerations for OSMR antibodies?

Species compatibility varies by antibody:

• Confirmed reactivity: The antibodies in the search results have demonstrated reactivity with human samples . Some also show reactivity with monkey samples .

• Predicted cross-reactivity: Based on sequence homology, human OSMR beta shares varying degrees of amino acid sequence identity with other species: 72% with canine, 61% with bovine, 58% with rat, and 55% with mouse OSMR beta . This suggests potential cross-reactivity, though validation is necessary.

• Domain considerations: Within the extracellular domain (ECD), human OSMR beta shares the homology percentages mentioned above with other species, which may impact antibody binding to specific domains .

• Functional studies across species: When studying receptor activation across species, note that the AB loop of OSM determines species-specific receptor activation patterns , which may affect functional studies even if antibodies cross-react.

• Validation requirement: Even for predicted reactive species, experimental validation is essential before use in critical experiments.

How can OSMR antibodies contribute to therapeutic development?

Emerging research indicates promising therapeutic applications:

• Reversing chemoresistance: Anti-OSMR human antibody treatment inhibits growth and metastasis of ovarian cancer cells and sensitizes cisplatin treatment, suggesting potential for combination therapy approaches .

• Targeting mechanism: OSMR fosters expression of specific integrin genes, creating a signaling crosstalk that contributes to cisplatin resistance. Blocking this pathway with OSMR antibodies represents a mechanistically-informed therapeutic strategy .

• Patient stratification: OSMR expression patterns could potentially help identify patients most likely to benefit from OSMR-targeted therapies or at risk for chemoresistance.

• Monitoring response: OSMR antibodies could be used to monitor receptor expression changes during treatment, potentially guiding therapeutic decisions.

What are the latest findings on OSMR's role in signaling pathways beyond STAT3?

While STAT3 is a primary mediator, OSMR activates multiple pathways:

• Additional STAT pathways: OSMR-containing receptors can potentially activate STAT1 and STAT5, with STAT5b specifically activated by OSMR but not other IL-6 family receptors .

• MAPK pathway: OSMR-containing receptors activate MAPK pathways in addition to JAK/STAT signaling .

• SHC activation: OSMR specifically activates SHC, which is not activated by other IL-6 family receptors .

• Integrin signaling crosstalk: OSMR regulates integrin gene expression, creating a signaling crosstalk that contributes to cancer cell behaviors like chemoresistance .

These diverse signaling outputs may explain the unique biological effects of OSMR activation compared to other cytokine receptors in the same family.

How do I select the most appropriate OSMR antibody for my specific research application?

Selection criteria should include:

• Application validation: Choose antibodies specifically validated for your application (WB, IHC, IF, flow cytometry, or ELISA) .

• Clone consideration: For monoclonal antibodies, specific clones may recognize different epitopes with varying functional implications (e.g., Clone # 469221) .

• Domain recognition: Consider whether the antibody recognizes the extracellular domain (e.g., Glu28-Ser739) or other regions relevant to your research question.

• Species compatibility: Verify reactivity with your species of interest, considering sequence homology if direct validation is unavailable .

• HRP conjugation needs: If direct detection is preferred, choose HRP-conjugated variants, but be aware this may limit flexibility in some experimental designs.

• Validation rigor: Prioritize antibodies validated in knockout systems for highest specificity confidence .

• Citation record: Consider antibodies with publication records in applications similar to yours (e.g., 10982-1-AP has been cited in multiple publications for WB, IHC, IF, and KD/KO studies) .

What future research directions are emerging for OSMR antibodies?

Based on current research trends:

• Combination therapy approaches: Further investigation of OSMR antibodies in combination with chemotherapy or other targeted therapies for cancer treatment .

• Receptor complex dynamics: More detailed study of OSMR dimerization and complex formation in different cellular contexts using advanced imaging and biochemical approaches .

• Biomarker development: Exploration of OSMR expression or activation as biomarkers for disease progression or treatment response.

• Humanized therapeutic antibodies: Development of humanized anti-OSMR antibodies for potential clinical applications based on promising preclinical results .

• Multi-omics integration: Combining OSMR antibody-based studies with genomic, transcriptomic, and proteomic analyses to develop more comprehensive understanding of OSMR biology in health and disease.