STAT1 Antibody

What is STAT1 and why is it important in cellular signaling research?

STAT1 (Signal Transducer and Activator of Transcription-1) is a member of the STAT family of cytoplasmic transcription factors that mediate cellular responses to cytokines, interferons, and growth factors. It plays a crucial role in signal transduction pathways, particularly those activated by interferons (IFNs) . Following Type I IFN (IFN-alpha and IFN-beta) binding to cell surface receptors, signaling via protein kinases leads to activation of Jak kinases (TYK2 and JAK1) and tyrosine phosphorylation of STAT1 and STAT2. The phosphorylated STATs dimerize and associate with ISGF3G/IRF-9 to form the ISGF3 transcription factor complex, which enters the nucleus and binds to IFN-stimulated response elements (ISRE) .

STAT1 is particularly important for research because:

• It regulates genes involved in cell growth, apoptosis, immune responses, and lipid metabolism

• It mediates cellular responses to various stimuli and pathogen exposure

• Loss of STAT1 has been implicated in the pathobiology of multiple cancer types

• It has distinct biological roles in different tissue contexts, including immune cells and mammary epithelium

How do I select the appropriate STAT1 antibody for my specific research application?

Selecting the appropriate STAT1 antibody requires careful consideration of several factors:

• Target epitope: Determine whether you need an antibody targeting specific domains or phosphorylation sites. Some antibodies recognize the N-terminal part (e.g., amino acids 69-169), while others target C-terminal regions (e.g., amino acids 650-750) .

• Application compatibility: Verify that the antibody has been validated for your specific application:

  • Western blot (WB) - Most antibodies are validated for this application

  • Immunohistochemistry (IHC) - Requires specific fixation compatibility

  • Immunofluorescence (IF/ICC) - Check cellular localization patterns

  • Flow cytometry - Verify antibody works in cell suspension conditions

  • ChIP applications - Ensure chromatin binding capability

• Host species and clonality: Consider whether you need a monoclonal antibody (for high specificity) or polyclonal (for robust detection):

  • Mouse monoclonal (e.g., clone 15H3 or 655210)

  • Rabbit polyclonal or monoclonal (e.g., EPR4407)

• Validation data: Review Western blot data showing specificity, ideally including STAT1 knockout cell lines as negative controls .

What is the difference between STAT1 isoforms, and how does this affect antibody selection?

STAT1 exists in two alternatively spliced isoforms: STAT1α (91 kDa) and STAT1β (84 kDa). The key differences relevant to antibody selection are:

• Structural differences: STAT1β lacks the C-terminal transactivation domain present in STAT1α .

• Functional implications: Both isoforms can be tyrosine-phosphorylated and form dimers, but STAT1β has reduced transcriptional activity due to the absence of the transactivation domain .

• Antibody selection considerations:

  • Antibodies targeting the N-terminus (before aa 687) will detect both STAT1α and STAT1β

  • Antibodies targeting the C-terminus (after aa 700) will preferentially detect STAT1α

  • RT-PCR analysis of STAT1 expression should consider both transcripts

In experimental contexts where distinguishing between isoforms is crucial, researchers should select antibodies that specifically target either shared regions (to detect total STAT1) or the C-terminal region (to detect only STAT1α) .

What are the optimal protocols for detecting STAT1 expression by Western blotting?

Western blotting is one of the most common applications for STAT1 antibodies. Based on established research protocols, here are the optimal conditions:

• Sample preparation:

  • Extract proteins using standard lysis buffers containing phosphatase inhibitors (critical for preserving phosphorylation status)

  • Use subcellular fractionation when distinguishing between cytoplasmic and nuclear STAT1

• Gel electrophoresis and transfer:

  • Use reducing conditions for most STAT1 antibodies

  • Expected band sizes: ~91 kDa (STAT1α) and ~84 kDa (STAT1β)

• Antibody incubation conditions:

  • Primary antibody dilutions typically range from 1:100 to 1:10,000, depending on the specific antibody

  • Some STAT1 antibodies require overnight incubation for optimal results

  • Use appropriate blocking buffer (5% non-fat dry milk in TBST is common)

• Data interpretation:

  • Verify band specificity using positive controls (e.g., IFN-treated cells like MCF7)

  • Use STAT1-knockout cell lines as negative controls when available

  • For quantification, normalize STAT1 band intensity to housekeeping proteins like β-actin

• Special considerations:

  • For phospho-STAT1 detection, stimulate cells with appropriate cytokines (IFN-α, IFN-γ) before lysis

  • Compare total STAT1 with phospho-STAT1 in parallel samples to assess activation status

How can I optimize STAT1 immunohistochemistry staining in tissue samples?

Immunohistochemistry (IHC) for STAT1 requires specific optimization steps:

• Tissue preparation and fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

  • Antigen retrieval is critical - heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is typically effective

• Staining protocol optimization:

  • Block endogenous peroxidase activity before antibody incubation

  • Use appropriate blocking serum to reduce background

  • Primary antibody dilutions typically range from 1:50 to 1:200 for IHC applications

  • Incubation times may require optimization (overnight at 4°C often yields best results)

• Signal interpretation and scoring:

  • Develop a consistent scoring system for STAT1 expression (example from research: sum of % of cells strongly positive × 3 and % of cells weakly positive × 1)

  • Consider both cytoplasmic and nuclear STAT1 staining separately, as they represent different activation states

  • Validate IHC findings with Western blot when possible

• Controls and validation:

  • Include known STAT1-positive tissues as positive controls

  • Use isotype controls to assess non-specific binding

  • Consider dual-staining with other markers to assess co-localization in specific cell types

What are the key considerations for detecting activated (phosphorylated) STAT1 versus total STAT1?

Detecting activated STAT1 requires specific approaches different from total STAT1 detection:

• Antibody selection:

  • Use phospho-specific antibodies targeting Tyr701 to detect IFN-activated STAT1

  • Use phospho-specific antibodies targeting Ser727 to detect additional activation events

  • Pair with total STAT1 antibodies in parallel samples for accurate interpretation

• Cell/tissue preparation:

  • For in vitro studies, stimulate cells with appropriate activators:

    • IFN-α or IFN-β for STAT1/STAT2 pathway activation

    • IFN-γ for STAT1 homodimer formation

    • KITLG/SCF for alternative activation pathways

  • Timing of stimulation is critical (typically 15-60 minutes)

  • Include phosphatase inhibitors in all buffers during sample preparation

• Experimental controls:

  • Unstimulated samples as negative controls

  • IFN-treated samples as positive controls

  • JAK inhibitor-treated samples to confirm specificity of phosphorylation

  • STAT1-knockout cell lines to validate antibody specificity

• Analytical approaches:

  • Co-immunoprecipitation to assess dimerization with STAT2 or other binding partners

  • Nuclear/cytoplasmic fractionation to assess nuclear translocation

  • Chromatin immunoprecipitation (ChIP) to assess DNA binding at target genes

How should I interpret STAT1 expression patterns across different cell types and tissues?

STAT1 expression varies considerably across cell types and tissues, requiring careful interpretation:

• Baseline expression patterns:

  • STAT1 is ubiquitously expressed but at varying levels

  • Immune cells typically show higher baseline expression

  • Cancer cell lines show variable expression (examples from research: STAT1 is detectable in EC1, KYSE150, KYSE510, NE2, NE3, and NE6 cell lines, while EC109 and SHEE were STAT1-negative)

• Subcellular localization interpretation:

  • Predominantly cytoplasmic staining indicates inactive STAT1

  • Nuclear localization suggests activated STAT1 involved in transcription

  • Both patterns may be present in responsive cells

• Context-dependent interpretation:

  • In cancer tissues, strong STAT1 expression often correlates with better differentiation

  • STAT1 expression in tumor stroma versus tumor cells may have opposing implications

  • STAT1 expression in ER-positive versus ER-negative breast tissues shows different patterns

• Comparative analysis approaches:

  • Use densitometry to quantify relative expression levels

  • Compare expression across multiple cell lines or tissues

  • Correlate expression with clinical or phenotypic parameters

What are common technical issues with STAT1 antibodies and how can I troubleshoot them?

Researchers frequently encounter technical challenges with STAT1 antibodies:

• High background or non-specific staining:

  • Increase blocking time or concentration

  • Optimize antibody dilution (typically start with manufacturer's recommendation, then test 2-fold dilutions)

  • Try alternative blocking agents (BSA vs. milk vs. serum)

  • Perform additional washing steps

• Weak or absent signal:

  • Verify STAT1 expression in your cell type (some cells express very low levels)

  • For Western blot, increase protein loading or exposure time

  • For IHC/IF, optimize antigen retrieval methods

  • Some antibodies require overnight incubation at 4°C for optimal results

  • Consider signal amplification methods

• Multiple bands in Western blot:

  • Expected bands are at 91 kDa (STAT1α) and 84 kDa (STAT1β)

  • Degradation products may appear as lower molecular weight bands

  • Phosphorylated forms may show slight mobility shifts

  • Cross-reactivity with other STAT family members can occur (verify with knockout controls)

• Inconsistent results between applications:

  • Not all antibodies work in all applications - verify validation data

  • Fixation conditions critical for IHC/IF may affect epitope recognition

  • Native vs. denatured protein recognition varies between antibodies

How can I validate the specificity of my STAT1 antibody results?

Validating antibody specificity is crucial for reliable research results:

• Genetic validation approaches:

  • STAT1-knockout cell lines provide the gold standard negative control

  • STAT1-knockdown cells using siRNA or shRNA can serve as partial controls

  • STAT1-overexpressing cells as positive controls

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be abolished or significantly reduced

  • Include non-competing peptide as control

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of STAT1

  • Consistent results with multiple antibodies increase confidence

  • Compare monoclonal and polyclonal antibody results

• Technical validation approaches:

  • Include proper positive and negative controls

  • For phospho-specific antibodies, include untreated and phosphatase-treated samples

  • For subcellular localization, verify with fractionation and Western blot

How can STAT1 antibodies be used to investigate the role of STAT1 in cancer progression?

STAT1 has complex roles in cancer that can be investigated using specific antibody-based approaches:

• Expression correlation studies:

  • Use IHC to correlate STAT1 expression with tumor characteristics:

    • In ESCC, STAT1 expression inversely correlates with tumor depth and size

    • Well/intermediate-differentiated tumors show stronger STAT1 immunostaining compared to poorly differentiated tumors

  • Compare expression in tumor versus stromal tissues

• Functional pathway analysis:

  • Detect phosphorylated STAT1 to assess pathway activation

  • Combine with downstream target detection (BCL-2, survivin, p21, cyclin D1)

  • Co-stain with other signaling molecules (STAT3, JAK kinases) to assess pathway crosstalk

• Prognostic value assessment:

  • Develop standardized scoring systems for STAT1 expression

  • Correlate expression patterns with clinical outcomes

  • Example study showed that in esophageal cancer, STAT1 expression inversely correlated with tumor depth and size (p = 0.047 and p = 0.029)

• Tumor microenvironment investigation:

  • Examine STAT1 in immune infiltrates versus tumor cells

  • Assess correlation with immune checkpoint markers

  • Study relationship with inflammatory cytokines in the tumor microenvironment

What methodological approaches can be used to study STAT1 deficiency and its biological consequences?

STAT1 deficiency has significant biological implications that can be studied through various methodologies:

• Models for studying STAT1 deficiency:

  • Complete recessive STAT1 deficiency (null alleles)

  • Partial dominant STAT1 deficiency (heterozygous mutations)

  • STAT1 knockout cell lines and animal models

  • Patient-derived cells with natural STAT1 mutations

• Functional assays for assessing STAT1 deficiency consequences:

  • IFN-responsive gene expression analysis

  • Cellular responses to pathogen challenges

  • DNA-binding activity assessment

  • Formation of bioactive γ-activating factor (GAF)

• Expression analysis in deficiency models:

  • RT-PCR to detect abnormal STAT1 transcripts

  • Western blotting to assess protein expression levels

  • In one study, cells from patients with partial STAT1 deficiency (P696S/P696S) showed approximately 10% of normal STAT1 protein levels

• Clinical correlation approaches:

  • Investigate susceptibility to mycobacterial diseases

  • Study responses to IFN therapies

  • Assess immune cell function in STAT1-deficient settings

How can I design experiments to study STAT1 interactions with other STAT family members and signaling molecules?

STAT1 functions within complex signaling networks that can be studied using specialized approaches:

• Co-immunoprecipitation studies:

  • Use STAT1 antibodies for immunoprecipitation followed by Western blot for interaction partners

  • Validated for MCF7 cells using anti-STAT1 antibody

  • Can detect interactions with STAT2, STAT3, ISGF3G/IRF-9, and other signaling molecules

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions in situ

  • Useful for detecting transient interactions following stimulation

  • Can be combined with subcellular localization studies

• ChIP and ChIP-seq approaches:

  • Study STAT1 binding to DNA targets

  • Compare binding patterns of different STAT family members

  • Identify cooperative or competitive binding sites

  • Several STAT1 antibodies have been validated for ChIP applications

• Multiplex immunofluorescence:

  • Simultaneously detect multiple STATs and signaling components

  • Assess co-localization in specific cellular compartments

  • Quantify relative expression levels in different cell types

• STAT1 phosphorylation dynamics:

  • Study temporal patterns of Tyr701 versus Ser727 phosphorylation

  • Investigate cross-regulation between different phosphorylation sites

  • Assess how phosphorylation affects interactions with other proteins

How can STAT1 antibodies be utilized in single-cell analysis techniques?

Single-cell technologies offer new opportunities for STAT1 research:

• Single-cell protein analysis:

  • Flow cytometry for phospho-STAT1 analysis in heterogeneous cell populations

  • Mass cytometry (CyTOF) to simultaneously analyze multiple STAT proteins and phosphorylation sites

  • Imaging mass cytometry for spatial resolution of STAT1 activation in tissues

• Spatial transcriptomics integration:

  • Combine STAT1 immunostaining with RNA expression profiling

  • Correlate STAT1 protein levels with target gene expression at single-cell resolution

  • Identify cellular niches with specific STAT1 activity patterns

• Multi-parametric analysis considerations:

  • Panel design for compatibility with other markers

  • Fixation and permeabilization optimization for intracellular detection

  • Quantitative analysis approaches for heterogeneous expression

What are the methodological considerations for studying STAT1 in the context of immune responses and immune cell function?

STAT1 plays critical roles in immune cells that require specific methodological approaches:

• Immune cell-specific protocols:

  • Stimulation conditions optimized for different immune cell types

  • Timing considerations for innate versus adaptive immune responses

  • Flow cytometry protocols for immune cell subpopulations

• Functional immune assays:

  • Cytokine production measurement in STAT1-sufficient versus STAT1-deficient cells

  • Antigen presentation and T cell activation studies

  • Pathogen clearance assays to assess innate immune function

• Tissue-specific immune microenvironment:

  • STAT1 expression in tumor-infiltrating lymphocytes

  • Comparison of STAT1 activity in stromal versus immune compartments

  • Research has shown that STAT1 in the immune system and surrounding mammary epithelial cells is centrally important for regulating tumor development

• Therapeutic response assessment:

  • STAT1 activation as a biomarker for immunotherapy response

  • Monitoring STAT1 phosphorylation during IFN therapy

  • Correlation with immune checkpoint expression

By addressing these comprehensive research questions with methodological rigor, researchers can effectively utilize STAT1 antibodies to advance understanding of this critical signaling molecule in normal physiology and disease states.