OAS3 Antibody, HRP conjugated

What is OAS3 and what cellular functions does it serve?

OAS3 (2'-5'-oligoadenylate synthetase 3) is an interferon-induced protein with a molecular weight of approximately 100-120 kDa that belongs to the 2-5A synthase family . It plays a critical role in antiviral action and signal transduction by catalyzing the synthesis of 2-prime,5-prime oligomers of adenosine (2-5As) . OAS3 is part of the larger 2′-5′-oligoadenylate synthetase family, which includes OAS1, OAS2, OAS3, and OASL . Research indicates that beyond its antiviral functions, OAS3 is associated with tumor progression and immune regulation, making it a significant target for cancer research .

What are the common applications for OAS3 antibody in research?

OAS3 antibody has multiple validated applications in research settings, each requiring specific optimization:

These applications allow researchers to investigate OAS3 expression in various experimental contexts, from protein level quantification to spatial localization in tissues . Research publications have documented successful use in knockdown/knockout validation, expression analysis in cancer tissues, and immune response studies .

Why is HRP conjugation useful for OAS3 antibody detection?

HRP (Horseradish peroxidase) conjugation provides significant advantages for OAS3 detection in research applications. As a 44 kDa glycoprotein with 6 lysine residues, HRP can be directly linked to antibodies to enable visualization through chromogenic reactions . The primary advantage of using HRP-conjugated OAS3 antibodies is direct detection, which eliminates cross-species reactivity concerns and reduces protocol time by removing additional wash and separation steps . This direct detection approach is particularly valuable in complex protocols involving multiple markers or when working with limited sample quantities.

What visualization methods work best with OAS3 antibody, HRP conjugated?

HRP conjugates can be visualized through several chromogenic reactions, each with distinct advantages:

• Diaminobenzidine (DAB) with hydrogen peroxide produces a water-insoluble brown pigment, ideal for long-term archiving of samples and counterstaining .

• ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) provides a soluble green end product suitable for quantitative ELISA applications .

• TMB (3,3',5,5'-tetramethylbenzidine) offers high sensitivity with a blue reaction product that turns yellow when stopped with acid, commonly used in ELISA and western blot applications .

• TMBUS offers enhanced sensitivity for detecting low abundance proteins like OAS3 in certain tissue contexts .

The choice of substrate should be determined by experimental requirements including detection sensitivity needs, archival importance, and counterstaining plans.

What are the optimal storage conditions for OAS3 antibody, HRP conjugated?

Proper storage is crucial for maintaining the functionality of OAS3 antibody with HRP conjugation. The recommended storage conditions are:

• Temperature: Store at -20°C for long-term preservation

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability: Stable for one year after shipment when stored properly

• Aliquoting: Not necessary for -20°C storage, but beneficial to avoid freeze-thaw cycles

• Dilution considerations: Performance diminishes over time, with accelerated degradation at higher temperatures and increased dilution

It's important to note that even with optimal storage, HRP conjugate performance gradually decreases over time, necessitating timely use and proper experimental controls .

What buffer considerations are important when working with OAS3 antibody, HRP conjugated?

The composition of the antibody buffer significantly impacts the performance of HRP-conjugated OAS3 antibody. Key buffer considerations include:

Importantly, buffers should not contain thiomersal/thimerosal, merthioloate, sodium azide, glycine, proclin, or nucleophilic components (primary amines like amino acids or ethanolamine, and thiols like mercaptoethanol or DTT) as these can significantly impair HRP activity or conjugation efficiency .

How should I validate the specificity of OAS3 antibody, HRP conjugated in my experimental system?

Rigorous validation of OAS3 antibody specificity is essential for meaningful research results. A comprehensive validation approach should include:

• Positive and negative control tissues/cells: Use tissues with known OAS3 expression (A375 cells, human placenta tissue, HeLa cells for WB; human skin cancer tissue, human brain tissue for IHC; A549 cells for IF/ICC)

• Knockdown/knockout validation: Multiple publications have demonstrated validation using OAS3 knockdown or knockout systems, providing a gold standard for specificity confirmation

• Western blot analysis: Confirm detection at the expected molecular weight (100-120 kDa observed for OAS3)

• Comparative analysis: Cross-validate results using alternative detection methods or antibodies targeting different epitopes of OAS3

• Reactivity assessment: Consider species-specific reactivity (tested in human samples, with cited reactivity in mouse samples)

For cancer-related research, validation in appropriate tissue contexts is particularly important given OAS3's differential expression across cancer types and stages .

What controls should be included when using OAS3 antibody, HRP conjugated?

Proper experimental controls are essential for interpreting results with OAS3 antibody, HRP conjugated:

• Positive tissue controls: Include samples with confirmed OAS3 expression such as A375 cells, HeLa cells, or human placenta tissue for western blot; human skin cancer or brain tissue for IHC; A549 cells for immunofluorescence

• Negative controls:

  • Isotype control: Use rabbit IgG at the same concentration as the OAS3 antibody

  • Secondary-only control: Omit primary antibody but include all other reagents

  • Antigen competition: Pre-incubate antibody with immunizing peptide (OAS3 fusion protein) to confirm binding specificity

• Expression manipulation controls:

  • OAS3 knockdown/knockout samples have been validated in multiple publications

  • Interferon-stimulated vs. non-stimulated samples (OAS3 is interferon-inducible)

• Technical controls:

  • Loading control for western blot (β-actin as used in published protocols)

  • Signal development time controls to prevent over-development

How can OAS3 antibody, HRP conjugated be used to study cancer progression?

Research has established OAS3 as a significant biomarker in cancer research, with HRP-conjugated antibodies enabling several important investigative approaches:

• Expression correlation with staging: OAS3 is aberrantly expressed in almost all TCGA cancer types and subtypes, with expression levels associated with tumor staging and metastasis . HRP-conjugated antibodies can quantify this expression through IHC and western blot analyses.

• Prognostic assessment: High OAS3 expression correlates with poor prognosis in multiple cancer types, including breast cancer . Researchers can use HRP-conjugated antibodies to:

  • Analyze expression in patient samples

  • Correlate expression with survival metrics (OS, DSS, DFS, PFS)

  • Develop tissue microarrays for high-throughput analysis

• Tumor microenvironment studies: OAS3 expression positively correlates with infiltration of immunosuppressive cells . HRP-conjugated antibodies enable co-localization studies with immune cell markers through sequential IHC or multiplex approaches.

• Treatment response prediction: OAS3 expression is associated with chemotherapeutic outcomes across various cancers . Researchers can use HRP-conjugated antibodies to evaluate pre- and post-treatment expression changes.

The methodology typically involves tissue sectioning, antigen retrieval (recommended with TE buffer pH 9.0 or citrate buffer pH 6.0), antibody incubation at optimized dilutions (1:20-1:200 for IHC), and visualization with appropriate HRP substrates .

What are the technical considerations for using OAS3 antibody in multiplexed detection systems?

Multiplexed detection involving OAS3 requires careful methodological planning:

• Sequential detection approach:

  • For HRP-conjugated OAS3 antibody used with other HRP-conjugated antibodies, complete stripping/blocking between rounds is essential

  • Consider using microwave treatment (10 mM citrate buffer, pH 6.0) or commercial stripping buffers between detections

  • Validate complete stripping by incubating with secondary antibody and substrate before proceeding

• Multi-fluorescence approach:

  • If converting from HRP to fluorescent detection, use tyramide signal amplification (TSA)

  • Carefully select fluorophores with non-overlapping spectra

  • When studying OAS3 in relation to immune infiltrates, consider established markers for immune cell populations as described in ImmuCellAI and TIMER2 databases

• Spectral unmixing considerations:

  • When using chromogenic substrates producing different colors, ensure proper image analysis algorithms

  • Consider tissue autofluorescence, particularly in tissues with high collagen content

• Co-localization analysis:

  • For studying OAS3 with DNA MMR genes, RNA methylation-related genes, and DNA methyltransferases, which show correlation with OAS3 expression , optimize antibody concentrations to achieve balanced signal intensity

How can OAS3 expression be quantified in research applications?

Quantification of OAS3 expression using HRP-conjugated antibodies can be approached through several methodologies:

• Western blot quantification:

  • Use β-actin as a normalization control as demonstrated in published protocols

  • Apply densitometry analysis to measure band intensity

  • Express results as percentages of normalized control signal following established procedures

• IHC scoring systems:

  • H-score (combines intensity and percentage of positive cells)

  • Allred score (particularly for cancer tissue analysis)

  • Digital image analysis using software that can distinguish between nuclear and cytoplasmic staining

• ELISA quantification:

  • Develop standard curves using recombinant OAS3 protein

  • Apply four-parameter logistic regression for concentration calculation

  • Consider multiplexed bead-based assays for analyzing OAS3 alongside other markers

• Integrative bioinformatics approaches:

  • Correlate protein expression with transcriptomic data

  • Analyze OAS3 in relation to immune infiltration scores (ImmuneScore, StromalScore, and ESTIMATEScore) using tools like the "ESTIMATE" R package

  • Compare OAS3 expression between tumor and adjacent normal tissues using standardized log2(TPM+1) values

What is the significance of OAS3 in studying interferon response and antiviral mechanisms?

OAS3 is a critical component of interferon-induced antiviral responses, and HRP-conjugated antibodies enable detailed studies of these mechanisms:

• Interferon stimulation experiments:

  • Treat cells with type I or type II interferons and quantify OAS3 induction

  • Analyze time-course responses to determine expression kinetics

  • Compare OAS3 induction across different cell types to identify tissue-specific responses

• Viral infection models:

  • Examine OAS3 upregulation following viral challenge

  • Correlate OAS3 expression with viral replication kinetics

  • Investigate the relationship between OAS3 levels and resistance to specific viral pathogens

• RNase L pathway analysis:

  • Study the relationship between OAS3-generated 2-5As and RNase L activation

  • Examine downstream RNA degradation patterns

  • Analyze cellular compartmentalization of the OAS3-RNase L axis during infection

• Immune signaling integration:

  • Investigate crosstalk between OAS3 and other interferon-stimulated genes

  • Study the relationship between OAS3 expression and patterns of immune cell infiltration

  • Examine how OAS3 expression correlates with neoantigen presentation in tumor contexts

How can I troubleshoot weak or inconsistent signal when using OAS3 antibody, HRP conjugated?

When encountering weak or inconsistent signals with OAS3 antibody, HRP conjugated, consider these methodological approaches:

• Antibody concentration optimization:

  • Perform a titration series across the recommended dilution range (1:1000-1:4000 for WB, 1:20-1:200 for IHC, 1:200-1:800 for IF/ICC)

  • If signal remains weak at highest recommended concentration, consider extending incubation time rather than further increasing concentration

• Antigen retrieval enhancement:

  • For IHC applications, compare TE buffer pH 9.0 with citrate buffer pH 6.0 as alternative retrieval methods

  • Optimize retrieval time and temperature for specific tissue types

  • For formalin-fixed tissues, extended retrieval may be necessary

• Signal amplification strategies:

  • Consider tyramide signal amplification (TSA) for substantial signal enhancement

  • Extend substrate incubation time (while monitoring background)

  • Use high-sensitivity HRP substrates like TMBUS for low-abundance detection

• Sample preparation refinement:

  • For western blot, ensure complete protein denaturation (5 min at 95°C in sample buffer)

  • Optimize protein loading amount (may need to increase for low-abundance samples)

  • Consider membrane type (PVDF may offer higher sensitivity than nitrocellulose for some applications)

• Storage and handling assessment:

  • Verify antibody storage conditions (-20°C, with glycerol and appropriate buffer)

  • Minimize freeze-thaw cycles

  • Check buffer composition for interfering components like sodium azide or high BSA content

How do experimental conditions affect OAS3 detection with HRP-conjugated antibodies?

Various experimental factors can significantly impact OAS3 detection:

• Cell/tissue treatment considerations:

  • Interferon stimulation dramatically increases OAS3 expression

  • Viral infection or TLR activation can induce significant expression changes

  • Cancer tissue may show aberrant expression patterns compared to normal tissue

• Fixation impact:

  • Formalin fixation time significantly affects epitope accessibility

  • Fresh frozen versus FFPE samples may require different antibody concentrations

  • For cell lines, paraformaldehyde (4%) versus methanol fixation may yield different results

• Buffer composition effects:

  • Ensure pH is within 6.5-8.5 range for optimal HRP activity

  • Avoid buffers containing >0.1% BSA or gelatin which can interfere with antibody binding

  • Eliminate sodium azide from all steps involving HRP detection as it inhibits peroxidase activity

• Incubation parameters:

  • Temperature (4°C overnight versus room temperature for shorter periods)

  • Agitation method (orbital shaker versus rocker)

  • Humidity considerations for IHC (maintain humid chamber to prevent section drying)

• Batch effects:

  • Different lots of antibody may require re-optimization

  • Include consistent positive controls across experiments to normalize for batch variation

What approaches can improve specificity when using OAS3 antibody, HRP conjugated?

Enhancing specificity requires multiple strategic approaches:

• Blocking optimization:

  • Compare different blocking agents (BSA, normal serum, commercial blockers)

  • Extended blocking periods (1-2 hours at room temperature or overnight at 4°C)

  • Include protein-free blockers when working with phosphorylated targets

• Wash protocol enhancement:

  • Increase wash buffer stringency (consider adding 0.1-0.3% Triton X-100)

  • Extend wash steps (5-10 minutes per wash, minimum 3 washes)

  • Use continuous gentle agitation during washing

• Antibody diluent considerations:

  • Addition of 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Include 1-5% normal serum from the same species as the tissue

  • Consider commercial antibody diluents specifically designed to reduce background

• Cross-reactivity reduction:

  • Pre-adsorption against tissue/cell lysates from negative control samples

  • For tissues with high endogenous biotin, use biotin blocking systems

  • Consider monovalent Fab fragments for secondary detection to reduce non-specific binding

• Validation through orthogonal methods:

  • Confirm findings using RNA expression data

  • Validate with knockdown/knockout controls

  • Compare with alternative antibody clones targeting different OAS3 epitopes

How is OAS3 antibody being used to study tumor immune microenvironment?

OAS3 antibody, HRP conjugated, has become an important tool for investigating the tumor immune microenvironment:

• Immune cell correlation studies:

  • Research has revealed that OAS3 expression positively correlates with infiltration of immunosuppressive cells

  • HRP-conjugated antibodies enable co-localization studies with immune cell markers

  • Sequential staining protocols can identify spatial relationships between OAS3-expressing cells and immune infiltrates

• Analytical approaches:

  • Integration with databases such as TIMER2, Xcell, CIBERSORT, and ImmuCellAI to analyze correlation between OAS3 expression and various immune cell types

  • Examination of 24 different immune cell types, including T cell subsets, using ImmuCellAI methodology

  • Application of XCell algorithm to examine multiple features of tumors based on gene expression data

• TME component analysis:

  • Assessment of tumor microenvironment composition using ImmuneScore, StromalScore, and ESTIMATEScore correlations with OAS3 expression

  • Investigation of OAS3's relationship with stromal and immune components within the tumor microenvironment

  • Spatial mapping of OAS3 expression relative to tumor boundaries and immune infiltration zones

• Therapeutic implications:

  • Analysis of OAS3 expression as a predictor of immunotherapy response using TIDE algorithm

  • Correlation of OAS3 levels with neoantigens in tumor samples

  • Investigation of OAS3 as a potential biomarker for immunotherapy patient selection

What role does OAS3 play in DNA repair mechanisms and how can this be studied?

Recent research has uncovered connections between OAS3 and DNA repair mechanisms:

• MMR pathway interactions:

  • OAS3 expression correlates with DNA mismatch repair (MMR) genes

  • HRP-conjugated antibodies can be used to study co-expression patterns in tissues

  • Western blot analysis can examine coordinate regulation of OAS3 with MMR components

• MSI relationship studies:

  • Defects in MMR lead to microsatellite instability (MSI)

  • Research approaches examine the relationship between OAS3 expression and MSI status

  • Correlation analyses between OAS3 levels and MMR protein expression in cancer samples

• Epigenetic regulation connections:

  • OAS3 expression shows relationships with DNA methyltransferases and RNA methylation-related genes

  • Methods include correlation analysis between OAS3 and genes involved in m6A, m1A, and m5C modifications

  • Examination of how epigenetic alterations affect OAS3 expression in different cancer contexts

• Therapeutic resistance implications:

  • Analysis of OAS3 expression in relation to drug sensitivity based on GDSC2 data

  • Investigation of the relationship between OAS3 levels and IC50 values for various therapeutics

  • Examination of how OAS3 status affects response to DNA damaging agents

How can OAS3 antibody be used to develop prognostic indicators in cancer research?

OAS3 has emerged as a potential prognostic biomarker, with HRP-conjugated antibodies enabling several research approaches: