OAS3 Antibody

What is OAS3 and what is its role in cellular function?

OAS3 is an interferon-induced, dsRNA-activated antiviral enzyme that plays a critical role in cellular innate antiviral responses. It belongs to the oligoadenylate synthetase family, which includes OAS1, OAS2, OAS3, and OASL. OAS3 functions by synthesizing 2'-5'-oligoadenylates (2-5A) from ATP in the presence of double-stranded RNA, which then bind to and activate ribonuclease L (RNase L). This activation leads to degradation of both cellular and viral RNA, inhibiting protein synthesis and terminating viral replication. Beyond its classical RNase L-dependent pathway, OAS3 can also mediate antiviral effects through alternative mechanisms and has demonstrated activity against various viruses including Chikungunya virus, Dengue virus, Sindbis virus, and Semliki forest virus .

What applications can OAS3 antibodies be used for in research?

OAS3 antibodies are versatile tools that can be employed in multiple experimental techniques:

Researchers should always validate antibodies in their specific experimental system, as performance can vary between different antibody clones and across applications .

What controls should be used when working with OAS3 antibodies?

For robust OAS3 antibody experiments, incorporate the following controls:

• Positive control: Use samples known to express OAS3, such as interferon-stimulated cells or tissues (A375 cells, HeLa cells, and human placenta tissue have been validated) .

• Negative control: Include samples where OAS3 expression is minimal or absent.

• Isotype control: Use an irrelevant antibody of the same isotype to assess non-specific binding.

• Blocking peptide control: Pre-incubate the antibody with its specific immunogen to confirm specificity.

• siRNA knockdown: Reduce OAS3 expression in cells to validate antibody specificity.

For western blotting, the predicted band size for human OAS3 is approximately 121 kDa . Discrepancies in band size may indicate post-translational modifications or alternative splicing variants.

How is OAS3 expression correlated with tumor immunity and cancer prognosis?

OAS3 has emerged as a significant co-immune biomarker associated with tumor progression and immune infiltration. Research has demonstrated that OAS3 is aberrantly expressed across almost all TCGA cancer types and subtypes, with expression levels correlating with tumor staging, metastasis, and prognostic deterioration in various cancers .

The relationship between OAS3 and tumor immune microenvironment (TME) is particularly noteworthy. OAS3 expression positively correlates with the infiltration of immunosuppressive cells, suggesting a potential role in immune evasion mechanisms. When investigating this relationship, researchers should consider:

• Analyzing OAS3 expression alongside ImmuneScore, StromalScore, and ESTIMATEScore using the "ESTIMATE" R package

• Utilizing platforms like TIMER2, Xcell, CIBERSORT, and ImmuCellAI to correlate OAS3 expression with specific immune cell infiltration

• Examining the association between OAS3 expression and immune checkpoint-related genes

• Investigating correlations between OAS3 expression and neoantigen load

This multi-dimensional analysis can provide insights into OAS3's role in cancer immunobiology and potential therapeutic implications.

What methodologies should be used to investigate OAS3's antiviral mechanisms?

Investigating OAS3's antiviral functions requires specialized approaches:

• Viral Infection Models: Establish cell culture systems infected with OAS3-sensitive viruses (CHIKV, Dengue, SINV, SFV) to assess the impact of OAS3 on viral replication.

• Pathway Analysis: Differentiate between RNase L-dependent and RNase L-independent antiviral mechanisms by:

  • Measuring 2-5A synthesis using HPLC or specialized assays

  • Assessing RNase L activation and RNA degradation patterns

  • Using RNase L knockout cells to identify alternative pathways

• Structure-Function Studies: Utilize antibodies targeting specific domains of OAS3 to elucidate the functional significance of its three OAS1-like domains.

• Metal Ion Dependency: Investigate how different metal ions (copper, iron, zinc, manganese) affect OAS3 enzymatic activity through in vitro enzymatic assays .

These approaches can provide comprehensive insights into OAS3's diverse antiviral mechanisms and potential therapeutic applications.

How can researchers investigate OAS3's role in the broader interferon response network?

OAS3 functions within a complex network of interferon-stimulated genes (ISGs). To understand its position in this network:

• Temporal Expression Analysis: Monitor OAS3 induction kinetics following interferon stimulation using qPCR and western blotting with validated antibodies.

• Interactome Mapping: Employ co-immunoprecipitation with OAS3 antibodies followed by mass spectrometry to identify protein-protein interactions.

• Signaling Pathway Integration: Use phospho-specific antibodies to examine how OAS3 influences or is influenced by key signaling nodes in interferon pathways.

• Transcriptomic Analysis: Perform RNA-seq in OAS3-overexpressing or OAS3-knockout models to identify downstream effectors and feedback mechanisms.

This systems biology approach can reveal how OAS3 coordinates with other ISGs to establish an antiviral state and influence cancer progression .

What are the optimal conditions for OAS3 Western blot analysis?

For successful OAS3 detection by Western blot:

• Sample Preparation:

  • Use RIPA buffer with protease inhibitors for cell lysis

  • Load 20-30 μg of total protein (as demonstrated with A431 whole cell lysate)

  • Include phosphatase inhibitors if studying OAS3 phosphorylation status

• Gel Electrophoresis:

  • Use 7.5% SDS-PAGE to properly resolve the 121 kDa OAS3 protein

  • Consider gradient gels (4-15%) for simultaneous detection of OAS3 and smaller proteins

• Transfer and Blocking:

  • Perform wet transfer for large proteins like OAS3 (>100 kDa)

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

• Antibody Incubation:

  • Primary antibody: Use at 1:1000-1:4000 dilution overnight at 4°C

  • Secondary antibody: HRP-conjugated at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • For weak signals, consider using signal enhancers or more sensitive detection reagents

What are the key considerations for OAS3 immunohistochemistry?

For optimal OAS3 detection in tissue sections:

• Tissue Processing:

  • Fixation: 10% neutral buffered formalin for 24-48 hours

  • Paraffin embedding following standard protocols

  • Section thickness: 4-5 μm

• Antigen Retrieval:

  • Primary method: TE buffer pH 9.0 (recommended)

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval using pressure cooker or microwave

• Antibody Selection and Dilution:

  • For human tissues: Use antibodies validated for human OAS3 at 1:20-1:200 dilution

  • For mouse/rat tissues: Select species-specific antibodies

• Detection Systems:

  • DAB (3,3'-diaminobenzidine) for brightfield microscopy

  • Fluorophore-conjugated secondary antibodies for fluorescence imaging

• Validated Positive Control Tissues:

  • Human skin cancer tissue

  • Human brain tissue

  • Human D54MG xenograft tissue

Researchers should optimize conditions based on their specific tissue type and fixation method.

How should researchers address batch-to-batch variability in OAS3 antibodies?

Antibody batch variation can significantly impact experimental reproducibility. To address this:

• Validation Strategy:

  • Test each new lot against a reference sample with known OAS3 expression

  • Compare titration curves between lots to establish equivalent working dilutions

  • Document lot numbers and validation data for publication

• Internal Standards:

  • Maintain aliquots of a standard positive control sample for lot comparison

  • Use recombinant OAS3 protein as a standardized control

• Multi-Antibody Approach:

  • When possible, confirm key findings with antibodies from different vendors or clones

  • Consider using antibodies targeting different epitopes of OAS3

• Detailed Record-Keeping:

  • Maintain a database of antibody performance across different experiments

  • Note any variations in detection sensitivity or specificity between lots

This systematic approach ensures experimental consistency despite inevitable batch variations.

Why might there be discrepancies in OAS3 detection patterns between antibodies?

Discrepancies in OAS3 detection can arise from multiple factors:

• Epitope Differences:

  • Different antibodies target distinct regions of OAS3 (e.g., some target within amino acids 50-400)

  • Epitope accessibility may vary depending on protein conformation or post-translational modifications

• Isoform Specificity:

  • OAS3 may exist in multiple isoforms or splice variants

  • Some antibodies may detect all isoforms while others are isoform-specific

• Cross-Reactivity:

  • Antibodies may cross-react with other OAS family members (OAS1, OAS2, OASL) due to sequence homology

  • Secondary cross-reactivity with unrelated proteins with similar epitopes

• Technical Variables:

  • Different fixation methods can affect epitope exposure

  • Sample preparation techniques may preserve or destroy certain epitopes

When discrepancies occur, researchers should validate findings with multiple detection methods and consider using genetic approaches (siRNA, CRISPR) to confirm specificity.

How can background issues be resolved when using OAS3 antibodies in immunofluorescence?

Background signal in OAS3 immunofluorescence can be minimized through these approaches:

• Antibody Optimization:

  • Titrate antibodies to find the optimal concentration (1:200-1:800 recommended)

  • Increase washing duration and frequency (4-5 washes of 5 minutes each)

• Blocking Enhancements:

  • Use image-specific blocking agents (normal serum from secondary antibody species)

  • Add 0.1-0.3% Triton X-100 for better antibody penetration

  • Include 0.1-1% BSA to reduce non-specific binding

• Fixation Considerations:

  • Compare paraformaldehyde, methanol, and acetone fixation to determine optimal epitope preservation

  • Adjust fixation time to minimize autofluorescence while preserving antigenicity

• Controls and Countermeasures:

  • Include a negative control without primary antibody

  • Use cells with OAS3 knockdown as a specificity control

  • Apply Sudan Black B (0.1-0.3%) to reduce autofluorescence

  • Consider spectral unmixing for multi-color experiments

These strategies have proven effective for OAS3 detection in A549 cells and other validated cell lines .

What factors affect OAS3 expression levels in experimental systems?

OAS3 expression is highly regulated and can be influenced by:

• Cytokine Stimulation:

  • Type I interferons (IFN-α, IFN-β) strongly induce OAS3 expression

  • Type II interferons (IFN-γ) may have cell type-specific effects

  • Pro-inflammatory cytokines can synergize with interferons

• Viral Infection:

  • Various RNA and DNA viruses trigger OAS3 upregulation

  • Virus-encoded antagonists may suppress OAS3 induction in certain infections

• Cell Type Differences:

  • Baseline and induced OAS3 expression varies substantially between cell types

  • Cancer cells may have dysregulated OAS3 expression

• Technical Considerations:

  • Cell culture conditions (confluency, passage number, serum factors)

  • RNA/protein extraction methods can affect yield and quality

  • Time-dependent expression patterns after stimulation

Researchers should standardize these variables and include appropriate time-course analyses when studying OAS3 regulation.

How can researchers integrate OAS3 data with broader immune pathway analyses?

Contextualizing OAS3 within immune networks requires:

• Multi-omics Integration:

  • Correlate OAS3 protein levels (antibody-based detection) with transcript data (RNA-seq)

  • Connect OAS3 expression with upstream regulators and downstream effectors

• Pathway Analysis Tools:

  • Use TIMER2, Xcell, CIBERSORT, and ImmuCellAI for immune infiltration correlation

  • Apply ESTIMATE algorithm for tumor microenvironment assessment

  • Employ Pearson correlation analysis for immune checkpoint gene associations

• Functional Validation:

  • Confirm computational predictions with knockout/knockdown experiments

  • Use OAS3 antibodies to monitor protein changes in response to pathway perturbations

• Therapeutic Relevance:

  • Correlate OAS3 expression with drug sensitivity using resources like GDSC2 and CTRP

  • Analyze immunotherapy response potential using the TIDE algorithm

This integrated approach provides a comprehensive understanding of OAS3's role in immune regulation and potential therapeutic applications.

How is OAS3 antibody research advancing our understanding of antiviral mechanisms?

OAS3 antibodies have enabled significant discoveries about antiviral immunity:

• Alternative Antiviral Pathways: Beyond the classical RNase L pathway, OAS3 has been found to mediate antiviral effects through RNase L-independent mechanisms, expanding our understanding of innate immune diversity .

• Virus-Specific Responses: OAS3 demonstrates varying efficacy against different viruses (CHIKV, Dengue, SINV, SFV), suggesting virus-specific interaction mechanisms that can be further explored using epitope-specific antibodies .

• Structural Insights: Antibodies targeting specific domains of OAS3 have helped elucidate how its three OAS1-like domains contribute to its unique antiviral properties compared to other OAS family members.

• Regulatory Networks: Immunoprecipitation studies with OAS3 antibodies have revealed previously unknown protein interactions that regulate OAS3 activity and localization.

These advances highlight the critical role of high-quality antibodies in deciphering complex antiviral mechanisms.

What are the implications of OAS3 in cancer immunotherapy research?

OAS3's emerging role in cancer biology opens new research avenues:

• Biomarker Potential: OAS3 expression correlates with tumor staging, metastasis, and prognostic deterioration across multiple cancer types, positioning it as a potential prognostic biomarker .

• Immune Checkpoint Interactions: OAS3 expression shows significant correlation with immune checkpoint genes, suggesting potential involvement in immunotherapy response mechanisms .

• Therapeutic Response Prediction: OAS3 expression patterns may help predict sensitivity to various chemotherapeutics and immunotherapies, as suggested by correlations with IC50 values in drug databases .

• Tumor Microenvironment Modulation: OAS3's association with immunosuppressive cell infiltration suggests it may influence the tumor immune microenvironment, affecting treatment outcomes .

Researchers investigating these aspects should employ validated antibodies in combination with genomic and transcriptomic approaches to comprehensively characterize OAS3's role in cancer immunity.