STAT4 Antibody, HRP conjugated

What is STAT4 and what cellular functions does it regulate?

STAT4 is a transcriptional regulator predominantly expressed in hematopoietic cells that plays critical roles in cellular growth, differentiation, and immune responses. It functions as a key mediator in the differentiation of T-helper 1 (Th1) cells and the production of interferon-gamma (IFN-γ) . After interleukin-12 (IL-12) binds to its receptor (IL12RB2), STAT4 interacts with the intracellular domain of IL12RB2 and undergoes tyrosine phosphorylation. The phosphorylated STAT4 molecules then homodimerize and translocate to the nucleus where they recognize specific STAT target sequences in IL-12 responsive genes . Beyond IL-12 signaling, STAT4 can also be activated by IFN-γ stimulation via JAK1 and TYK2, as well as by other interleukins including IL-23, IL-2, and IL-35 . Additionally, STAT4 participates in neutrophil functions such as chemotaxis and the production of neutrophil extracellular traps .

How do HRP-conjugated antibodies enhance STAT4 detection sensitivity?

HRP-conjugated antibodies provide significant advantages for STAT4 detection through enzymatic signal amplification. The horseradish peroxidase enzyme catalyzes reactions with chromogenic, fluorogenic, or chemiluminescent substrates, generating visual signals that can be detected at very low protein concentrations. When using high-sensitivity ECL substrates, HRP-conjugated antibodies enable detection of proteins in the mid-femtogram range . For STAT4 detection, this enzymatic amplification is particularly valuable in tissues or cells where expression levels may be heterogeneous or relatively low. The signal amplification property of HRP conjugation allows researchers to visualize STAT4 expression patterns in complex tissues like thymus or tumors where detection might otherwise be challenging without signal enhancement .

What are the most reliable applications for STAT4 antibody, HRP conjugated?

HRP-conjugated STAT4 antibodies demonstrate reliability across multiple immunodetection techniques. Western blotting applications consistently yield distinct bands at the predicted 85 kDa size for STAT4 protein, with minimal background interference when using optimized blocking conditions (5% NFDM/TBST) . Immunoprecipitation procedures show high specificity, successfully isolating STAT4 from complex tissue lysates such as thymus tissue . For immunohistochemistry, HRP-conjugated STAT4 antibodies provide clear cellular localization data in paraffin-embedded tissues with minimal background staining when incubated at appropriate dilutions (1/500 concentration) and appropriate incubation times (30 minutes at room temperature) . For highest reliability, verification with positive controls (like lymphoid tissues) and negative controls (isotype-matched antibodies) should be incorporated into experimental design.

What validation methods ensure STAT4 antibody specificity?

Validating STAT4 antibody specificity requires a multi-parameter approach. Western blot analysis should consistently detect a single band at the predicted 85 kDa molecular weight across different sample types . Immunoprecipitation followed by western blotting (IP-WB) provides critical confirmation of specificity by demonstrating the antibody's ability to isolate the target protein from complex mixtures and subsequently detect it . Knockout/knockdown validation, though not explicitly mentioned in the search results, represents the gold standard for antibody validation—comparing detection in wild-type samples versus those where STAT4 expression has been genetically eliminated or reduced. Peptide competition assays can further confirm binding specificity by demonstrating signal reduction when the antibody is pre-incubated with its target peptide. Cross-reactivity testing against other STAT family members, particularly the structurally similar STAT1, is essential for confirming STAT4-specific detection.

How can STAT4 antibodies be optimized for detecting phosphorylated versus total STAT4?

Detecting phosphorylated STAT4 requires careful consideration of epitope accessibility and phosphorylation-induced conformational changes. For optimal detection of phosphorylated STAT4, researchers should:

• Select antibodies specifically raised against phospho-epitopes (typically pY693 for STAT4) that do not cross-react with non-phosphorylated forms.

• Incorporate phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate) in all sample preparation buffers to preserve phosphorylation status.

• Optimize cell stimulation conditions with IL-12, as the search results indicate this cytokine is the predominant activating signal for STAT4 phosphorylation through the IL-12R/JAK2 signaling pathway .

• Consider dual staining approaches that employ separate antibodies for total STAT4 and phospho-STAT4 to calculate activation ratios.

• Implement rapid sample processing protocols, as phosphorylation states can be transient and susceptible to degradation by endogenous phosphatases.

When comparing phosphorylated versus total STAT4 levels, normalization against total STAT4 provides more informative data about pathway activation states than simple phospho-STAT4 quantification alone.

What methodological approaches optimize STAT4 detection in breast cancer research?

Breast cancer research presents unique challenges for STAT4 detection due to expression heterogeneity across different cell populations. Single-cell transcriptomics analyses of breast cancer cohorts reveal that STAT4 expression varies significantly among patients and is predominantly expressed in tumor-infiltrating immune cells rather than malignant or stromal cells . To optimize detection methods:

• Implement dual immunohistochemistry to simultaneously visualize STAT4 and cell-type markers (CD4, CD8) to accurately interpret expression patterns.

• Consider tissue microarray approaches to account for intratumoral heterogeneity, as STAT4 levels vary across different regions of breast tumors.

• Integrate single-cell analysis techniques alongside bulk tissue measurements to deconvolute the cell-type specific expression of STAT4 in complex tumor samples .

• Correlate STAT4 protein expression with T-cell infiltration metrics, as research demonstrates a strong positive correlation (Cor = 0.91, p-value < 0.001) between T-cell ratio and STAT4-positive cell ratio in primary tumors .

• Employ multiplex immunofluorescence when available to simultaneously detect STAT4 with STAT3, PD-L1, and IL-12 receptor components to validate the proposed IL-12R/JAK2–STAT3–STAT4/PD-L1 feedback loop .

What factors influence STAT4 antibody performance in different experimental contexts?

Multiple factors can significantly impact STAT4 antibody performance across different experimental applications:

For optimal results, each parameter should be systematically optimized for the specific tissue type and experimental question being investigated.

How can STAT4 antibodies contribute to understanding immunotherapy response prediction?

STAT4 antibodies provide critical tools for investigating immunotherapy response mechanisms and developing predictive biomarkers. Research has established that STAT4 expression correlates with anti-PD-1 immunotherapy response through several mechanisms:

• STAT4 facilitates PD-L1 expression via the IL-12R/JAK2/STAT3 axis, creating a feedback loop (STAT4/IL-12R/JAK2–STAT3–STAT4/PD-L1) that influences immunotherapy response .

• The STAT4-related pathway score (Srps), calculated using gene expression levels of JAK2, STAT3, STAT4, CD274 (PD-L1), IL12RB1, and IL12RB2, demonstrates higher predictive accuracy (AUC = 0.83) for anti-PD-1 treatment response compared to other biomarkers .

• STAT4 and Srps are significantly elevated after anti-PD-1 treatment, with responding patients showing higher expression levels .

• Strong correlations exist between STAT4 expression and T-cell infiltration, particularly CD4+ T cells, CD8+ T cells, and NK cells (correlation coefficients > 0.8, p-value < 0.001) .

Researchers can employ STAT4 antibodies in multiplexed IHC panels to assess these relationships in clinical samples, potentially developing companion diagnostic approaches for patient stratification in immunotherapy trials.

What technical considerations are important when using STAT4 antibody in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with STAT4 antibodies requires careful experimental design to preserve protein-protein interactions and minimize artifacts:

• Lysate preparation: Use gentle lysis buffers (typically containing 1% NP-40 or 0.5% Triton X-100) to preserve native protein complexes, particularly the STAT3-STAT4 interaction verified through Co-IP .

• Antibody selection: Choose STAT4 antibodies validated specifically for immunoprecipitation applications, such as those demonstrated effective at 1/30 dilution (2μg in 0.35mg lysates) .

• Controls: Always include isotype control antibodies (e.g., rabbit monoclonal IgG) to identify non-specific binding .

• Detection system: For western blot analysis of immunoprecipitated material, use specialized secondary antibodies like VeriBlot for IP Detection Reagent to minimize detection of the immunoprecipitating antibody's heavy and light chains .

• Verification of interactions: Given that STAT4 interacts with STAT3 in the context of PD-L1 regulation, reciprocal Co-IPs (immunoprecipitating with anti-STAT3 and detecting STAT4, and vice versa) provide stronger evidence for specific interactions .

• Interpretation: Be aware that degradation bands may appear (as noted in the lower band in Co-IP results) , requiring careful analysis to distinguish between degradation products and actual interaction partners.

How can STAT4 antibodies be integrated into studies of JAK2/STAT3/PD-L1 signaling in cancer?

STAT4 antibodies serve as essential tools for dissecting the complex JAK2/STAT3/PD-L1 signaling network in cancer research through several methodological approaches:

• Combinatorial knockdown studies: Using STAT4 antibodies to monitor protein levels following siRNA-mediated silencing of pathway components helps establish dependency relationships, as demonstrated in studies showing that STAT3 silencing reduces CD274 (PD-L1) levels induced by STAT4 overexpression .

• Chromatin immunoprecipitation (ChIP): Apply STAT4 antibodies in ChIP assays to identify direct transcriptional targets, particularly IL12RB1 and IL12RB2, which were confirmed as STAT4-regulated genes .

• Pathway visualization: Implement multiplexed immunofluorescence with antibodies against STAT4, STAT3, phospho-JAK2, and PD-L1 to visualize the spatial relationships of these proteins in tissue samples.

• Therapeutic response monitoring: Utilize STAT4 antibodies to track changes in the STAT4-related pathway following treatments that target this signaling axis, since components of this pathway are increased following anti-PD-1 therapy .

• Translational applications: Apply the STAT4-related pathway score (Srps) as a potential stratification tool for immunotherapy selection, as higher Srps correlates with "hot" tumor microenvironments and better immunotherapy response .

Research indicates this approach is particularly valuable in breast cancer, where the STAT4-related pathway shows promise for predicting response to anti-PD-1 immunotherapy.

How should researchers address non-specific background when using STAT4 antibody, HRP conjugated?

Non-specific background represents a common challenge when using HRP-conjugated antibodies. To minimize this issue with STAT4 antibody detection:

• Optimize blocking conditions: 5% non-fat dry milk in TBST has been demonstrated effective for STAT4 antibody applications . Alternative blocking agents like bovine serum albumin (BSA) or casein may be tested if milk proteins interact with the sample.

• Adjust antibody dilution: Titrate the primary antibody concentration to identify the optimal signal-to-noise ratio. For IHC applications, 1/500 dilution (1.136 μg/ml) has been validated as effective .

• Optimize incubation parameters: Reduce incubation temperature (4°C overnight instead of room temperature) or extend washing steps to reduce non-specific binding.

• Consider detection systems: For IHC applications, polymer-based detection systems like Leica DS9800 (BOND™ Polymer Refine Detection) demonstrate good specificity with minimal background .

• Incorporate additional blocking steps: When working with tissues rich in endogenous biotin or peroxidase activity, include specific blocking steps (avidin/biotin block or peroxidase quenching) prior to antibody application.

• Evaluate alternative secondary antibodies: If using indirect detection methods, select secondary antibodies with minimal cross-reactivity to human IgG to reduce background in human samples .

What approaches help resolve contradictory STAT4 expression data between different detection methods?

Researchers frequently encounter seemingly contradictory STAT4 expression data when comparing results from different methodologies. To reconcile these discrepancies:

• Recognize cell type specificity: STAT4 expression shows significant heterogeneity across cell types, being predominantly expressed in immune cells rather than malignant or stromal cells in cancer samples . Flow cytometry or single-cell analysis may detect STAT4 in specific immune populations that are diluted in bulk tissue analysis.

• Consider subcellular localization: STAT4 shuttles between cytoplasm and nucleus upon activation, resulting in different detection patterns depending on fixation methods and the epitope recognized by the antibody.

• Account for post-translational modifications: Phosphorylation status significantly affects STAT4 detection with certain antibodies. Ensure the antibody's epitope specificity is compatible with the biological state being studied.

• Evaluate detection sensitivity thresholds: Different methods have varying sensitivity limits. Western blotting with ECL detection can detect femtogram quantities, while IHC may require higher expression levels .

• Analyze expression dynamics: Single-cell transcriptomics data reveal that STAT4 expression varies significantly among patient cohorts, showing different patterns across cell clusters . This natural heterogeneity explains some apparent contradictions.

• Verify with orthogonal methods: When conflicting data emerge, implement multiple detection techniques (protein, mRNA, functional assays) to build a consensus understanding of STAT4 biology in the system.

What strategies optimize STAT4 antibody performance in multiplex immunofluorescence applications?

Optimizing STAT4 antibodies for multiplex immunofluorescence requires addressing several technical considerations:

• Antibody panel design: When designing panels to study the STAT4-related pathway, carefully select antibodies raised in different host species to avoid cross-reactivity. For studying the IL-12R/JAK2–STAT3–STAT4/PD-L1 axis, ensure antibodies targeting each component are compatible.

• Signal amplification: For low-abundance targets, consider tyramide signal amplification (TSA) to enhance detection while allowing antibody stripping and sequential staining.

• Epitope retrieval optimization: Different antibodies in a multiplex panel may require different antigen retrieval conditions. Perform comprehensive testing to identify a universal retrieval protocol that preserves all epitopes of interest.

• Spectral unmixing: Implement spectral imaging and computational unmixing to separate overlapping fluorophore signals, particularly important when studying multiple STAT pathway components simultaneously.

• Sequential staining: For complex panels, employ sequential staining with antibody stripping between rounds rather than simultaneous application of all antibodies.

• Validation with single-stain controls: Always include single-stain controls to verify antibody performance in the multiplex context and confirm appropriate spectral unmixing.

These approaches are particularly valuable for investigating the relationships between STAT4, T-cell markers, and components of the PD-L1 pathway in cancer samples, where understanding cellular co-localization provides critical insights into immune regulation .

How can STAT4 antibodies contribute to understanding immunotherapy resistance mechanisms?

STAT4 antibodies provide valuable tools for investigating mechanisms of immunotherapy resistance through several research approaches:

• Comparative expression analysis: Apply STAT4 antibodies to compare expression patterns between responsive and resistant tumors. Research demonstrates that STAT4 and its related pathway components show higher expression in responders to anti-PD-1 therapy compared to non-responders .

• Dynamic response monitoring: Use sequential biopsies analyzed with STAT4 antibodies to track changes during treatment, as studies show STAT4-related pathway components increase following anti-PD-1 therapy in responding patients .

• Functional correlation studies: Combine STAT4 detection with quantification of T-cell functionality markers, as STAT4 expression strongly correlates with T-cell expansion following immunotherapy .

• Mechanistic intervention studies: Apply STAT4 antibodies to monitor pathway modulation following experimental interventions targeting the IL-12R/JAK2/STAT3 axis, which may overcome resistance by enhancing STAT4 activity.

• Predictive biomarker development: Incorporate STAT4 antibodies in multiplexed panels to validate the STAT4-related pathway score (Srps) as a predictive biomarker, which shows superior accuracy (AUC = 0.83) compared to other immune biomarkers .

These approaches leverage STAT4's role in regulating PD-L1 expression through the IL-12R/JAK2–STAT3–STAT4/PD-L1 feedback loop, potentially identifying targetable mechanisms to overcome immunotherapy resistance.

What are the possibilities for developing STAT4-targeted therapeutic antibody conjugates?

The development of STAT4-targeted therapeutic antibody conjugates represents an emerging research direction with several considerations:

• Targeting strategy: Since STAT4 functions intracellularly, therapeutic approaches must overcome membrane barriers. Antibody-drug conjugate (ADC) technologies like the pair-FORCE system could potentially deliver payloads to cells expressing surface markers associated with high STAT4 activity .

• Cell-type selectivity: STAT4 shows specific enrichment in T cells within the tumor microenvironment , suggesting that STAT4-modulating therapies could selectively enhance anti-tumor immune responses without directly targeting cancer cells.

• Conjugation considerations: Modern site-specific conjugation approaches would be essential for developing STAT4-related therapeutics, as heterogeneous conjugation can result in reduced stability, poor pharmacokinetics, decreased tumor penetration, and increased systemic toxicity .

• Functional potentiation: Rather than directly targeting STAT4, therapeutic antibodies could target upstream pathway components like IL-12 receptors to enhance STAT4 activation, potentially converting "cold" tumors to "hot" immunologically active tumors.

• Combination approach: STAT4 agonism could be particularly valuable as an immune induction strategy prior to or concurrent with PD-1 blockade therapy, as STAT4 overexpression promotes PD-L1 expression via the JAK2/STAT3 signaling axis .

Experimental evidence suggests that activation of STAT4 may promote favorable tumor microenvironment conditions for cancer immunotherapy, making this pathway an attractive target for therapeutic development .

How might STAT4 antibodies be applied in single-cell analysis technologies?

STAT4 antibodies can be integrated into cutting-edge single-cell analysis technologies through several methodological approaches:

• Mass cytometry (CyTOF): Conjugate STAT4 antibodies with rare earth metals for high-dimensional protein analysis at single-cell resolution, allowing simultaneous detection of STAT4 with other signaling molecules and lineage markers.

• Single-cell western blotting: Apply STAT4 antibodies in microfluidic single-cell western blotting platforms to quantify protein levels in individual cells, revealing heterogeneity masked in bulk analyses.

• Imaging mass cytometry: Use metal-conjugated STAT4 antibodies for high-multiplexed tissue imaging, preserving spatial relationships between STAT4-expressing cells and their microenvironment.

• CITE-seq: Employ oligonucleotide-tagged STAT4 antibodies in cellular indexing of transcriptomes and epitopes by sequencing, correlating STAT4 protein expression with transcriptome-wide changes at single-cell resolution.

• Spatial transcriptomics integration: Combine STAT4 antibody-based protein detection with spatial transcriptomics to map the relationship between STAT4 protein expression and gene expression programs in preserved tissue architecture.

These approaches address the significant heterogeneity of STAT4 expression observed in single-cell transcriptomics datasets, where STAT4 shows variable expression patterns across different cohorts and cell clusters . Such technologies can help understand how STAT4 coordinates immune responses in complex tissues where cell-cell interactions are critical for function.

What are the key considerations for standardizing STAT4 detection across research laboratories?

Standardizing STAT4 detection requires addressing several technical and methodological variables:

• Antibody validation: Establish consensus validation criteria, including western blot confirmation of specificity at the expected 85 kDa molecular weight, verification through immunoprecipitation, and testing in knockout/knockdown systems .

• Protocol standardization: Develop detailed standard operating procedures for sample preparation, including optimal fixation methods for tissue samples and lysis conditions for protein extraction.

• Reporting standards: Implement comprehensive reporting requirements including antibody catalog numbers, dilutions, incubation conditions, detection systems, and positive and negative controls used in experiments.

• Reference materials: Establish common reference samples with validated STAT4 expression levels to enable inter-laboratory calibration.

• Algorithm standardization: For quantitative analyses, particularly in digital pathology applications, develop consensus algorithms for measuring STAT4 expression and calculating derived metrics like the STAT4-related pathway score .

• Cross-platform validation: Perform systematic comparisons of STAT4 detection across different platforms (western blot, IHC, flow cytometry) to establish expected concordance patterns and explain predictable discrepancies.

These standardization efforts would significantly enhance data reproducibility and facilitate development of STAT4-based biomarkers for clinical applications such as predicting immunotherapy response.

How might future advances in antibody engineering improve STAT4-targeted research tools?

Emerging antibody engineering technologies hold significant promise for developing next-generation STAT4 research tools:

• Recombinant antibody fragments: Development of smaller antibody formats (Fab, scFv, nanobodies) against STAT4 could improve tissue penetration for imaging applications and enable access to cryptic epitopes.

• Bifunctional antibodies: Engineering antibodies that simultaneously bind STAT4 and interacting partners like STAT3 could enable selective detection of functional complexes rather than individual proteins .

• Phospho-state selective binders: Advanced selection strategies could yield antibodies that selectively recognize different phosphorylation states of STAT4, providing direct readouts of activation status.

• Intracellular antibody delivery: Developing technologies for delivering antibodies into live cells could enable real-time monitoring of STAT4 nuclear translocation and complex formation during signaling events.

• Proximity-based reporters: Engineering split reporter systems fused to anti-STAT4 antibody fragments could enable real-time visualization of STAT4 dimerization or interaction with other pathway components.

• Format Chain Exchange (FORCE) technology adaptation: Implementing approaches similar to the pair-FORCE system described for bispecific antibodies could enable efficient generation of diverse STAT4 antibody-payload conjugates for research applications .