STAT4 Antibody, Biotin conjugated
Description
Definition and Mechanism
STAT4 Antibody, Biotin Conjugated, is a biotinylated monoclonal antibody designed to detect the phosphorylated or non-phosphorylated forms of STAT4, a transcription factor critical in immune regulation. Biotin conjugation enables its use in detection systems involving streptavidin-enzyme complexes, enhancing sensitivity in assays like Western blotting, immunohistochemistry (IHC), and ELISA.
| Feature | Details |
|---|---|
| Target | Signal transducer and activator of transcription 4 (STAT4) |
| Conjugate | Biotin (via NHS-ester chemistry for covalent attachment) |
| Applications | Western blot, flow cytometry, IHC, immunoprecipitation (IP) |
| Reactivity | Human, mouse, rat (species-specific validation required) |
| Immunogen | Full-length recombinant STAT4 protein or synthetic peptides |
Role in Immune Cell Signaling
STAT4 antibodies are critical for studying STAT4’s activation in response to IL-12, IL-23, and IFN-γ. Key findings include:
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Th1/Th17 Differentiation: STAT4-deficient mice exhibit impaired Th1/Th17 responses and heightened Th2 activity, exacerbating autoimmune diseases like experimental autoimmune encephalomyelitis (EAE) .
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Macrophage and Dendritic Cell Function: STAT4 is induced during maturation of these antigen-presenting cells (APCs), enabling autocrine IL-12 signaling and IFN-γ production . Th2 cytokines (e.g., IL-4) suppress STAT4 expression, modulating APC-mediated immune responses .
NK Cell Activation
STAT4 antibodies reveal elevated basal STAT4 levels in NK cells, enabling rapid IFN-γ production during viral infections. In Toxoplasma gondii infection, STAT4-deficient macrophages fail to produce nitric oxide (NO) and exhibit reduced pathogen clearance .
Isoform-Specific Regulation
STAT4 exists in two isoforms:
| Isoform | Characteristics | Functional Impact |
|---|---|---|
| STAT4α | Full-length; promotes IL-10 production | Attenuates EAE severity; anti-inflammatory |
| STAT4β | Lacks 44 C-terminal amino acids; enhances IFN-γ/IL-17 production | Exacerbates EAE; pro-inflammatory |
Transgenic mice expressing STAT4β develop severe EAE compared to STAT4α or wild-type mice, highlighting isoform-specific roles in cytokine regulation .
Detection Methods
| Method | Protocol Highlights | Optimal Dilutions |
|---|---|---|
| Western Blot | Denaturing SDS-PAGE; membrane blocking with 5% BSA; detection via streptavidin-HRP | 1:500–1:1000 |
| Flow Cytometry | Fixation/permeabilization required; intracellular staining with biotin-streptavidin conjugates | 1:100–1:200 |
| IHC | Paraffin-embedded sections; antigen retrieval (e.g., heat-induced epitope retrieval) | 1:50–1:200 |
Example Workflow:
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Western Blot: Load lysates (40 µg/lane); probe with biotin-STAT4 antibody (1:500) followed by streptavidin-HRP and ECL detection .
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Immunoprecipitation: Use anti-STAT4 antibody to pull down STAT4 complexes for downstream phosphatase assays .
Critical Considerations
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Cross-Reactivity: Ensure species-specific validation (e.g., human vs. mouse STAT4) .
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Phospho-Specific Variants: For detecting activated STAT4 (e.g., pY693), use non-conjugated phospho-specific antibodies (e.g., Boster A00734Y693) .
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Control Experiments: Include isotype-matched controls (e.g., biotin-conjugated IgG) to rule out non-specific binding .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- This study proposes that variations in the GATA3 transcription factor, rather than STAT4, are associated with the risk of type 2 diabetes in the Bangladeshi population. PMID: 30044774
- Despite the association of STAT4 gene SNPs (rs7574865 and rs7601754) with the risk of adult Systemic Lupus Erythematosus (SLE) in the Iranian population, they are not associated with Juvenile Systemic Lupus Erythematosus (JSLE). This suggests differences in the genetic background of JSLE and SLE. PMID: 29276866
- No significant relationships were found between STAT4 variants and serum neopterin or disease activity parameters. The study confirmed the association of STAT4 rs7574865 polymorphism with Rheumatoid Arthritis (RA) and was the first to identify an association with Rheumatoid Factor and anti-cyclic citrullinated peptide antibodies positivity. PMID: 28424905
- Five STAT4 SNPs were analyzed in 233 patients with established Neuromyelitis Optica Spectrum Disorder (NMOSD) and 492 healthy controls. Allelic, additive, dominant, and recessive models identified associations with NMOSD. Minor alleles of four STAT4 SNPs exhibited significant association with increased risk of NMOSD: rs7574865 T; rs10181656 G; rs10168266 T; and rs13426947 A in all models. rs7601754 G displayed a protective effect against NMOSD. PMID: 28852993
- Data demonstrate that STAT4 plays a role in enabling the normal and timely division of cells undergoing mitosis. PMID: 28516569
- A genome-wide association study identified a locus in STAT4, rs13390936, associated with nontyphoidal Salmonella bacteremia. PMID: 29523850
- Neither alleles nor genotypes of rs7601754 SNP of the STAT4 gene demonstrated associations with Juvenile Rheumatoid Arthritis (JRA). This study found that gene variants of STAT4 did not affect JRA susceptibility in an Iranian population. PMID: 28524764
- STAT4 is a novel transcriptional regulator of p66Shc in normal and chronic lymphocytic leukemia B cells. PMID: 27494881
- IL13 AA of rs20541 and STAT4 TT of rs925847 are potential genomic biomarkers for predicting lower pulmonary function. Administering high-dose inhaled corticosteroids (ICSs) to asthmatic patients with genetic variants of IL13 AA may inhibit the progression of airway remodeling. The genetic variants of STAT4 TT did not respond to high-dose ICSs. PMID: 26765219
- STAT4 mRNA expression was significantly correlated with IFNG mRNA expression. PMID: 29187449
- Findings demonstrate that variants in STAT4 play a critical role in Hepatitis B virus (HBV) infection and clearance in the Chinese Han population. PMID: 27444301
- STAT4 rs7574865G/T polymorphism is associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus in Mexican women. PMID: 27178308
- STAT4_rs7574865 TT was associated with the presence of actively inflamed joints and extra-articular damage in patients with Juvenile Idiopathic Arthritis. PMID: 28145159
- Results indicate that activated STAT4 is overexpressed in epithelial cells of ovarian cancer and provide evidence that it promotes ovarian cancer metastasis via tumor-derived Wnt7a-induced activation of cancer-associated fibroblasts. PMID: 28114283
- STAT4 rs7574865 gene polymorphism is associated with the susceptibility of primary biliary cirrhosis in the Han population of Jiangsu province. PMID: 28395724
- The findings confirm the important role of the STAT4 gene in the predisposition to Systemic Sclerosis and its phenotypes, such as the presence of Interstitial Lung Disease, cardiac injury, and seropositivity for anti-topoisomerase I antibodies in the Russian population. PMID: 28631694
- Results show a specific and dominant contribution of STAT4 in the hematopoietic compartment to metabolic health and inflammation in diet-induced obesity. PMID: 28400678
- This study reported that SNPs in STAT4, PTPN2, PSORS1C1, and TRAF3IP2 are associated with response to TNF-i treatment in RA patients; however, these findings should be validated in a larger population. PMID: 28107378
- This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-alpha/beta signaling, highlighting the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells. PMID: 26990433
- A higher risk of developing RA was observed for rs7574865 in the STAT-4 gene, while the rs1800872 in the IL-10 gene showed a protective effect. PMID: 27342690
- STAT4 rs7574865 polymorphism has a role in Rheumatoid Arthritis and disease activity, but not in anti-CCP antibody levels in a Mexican population. PMID: 27234231
- This study demonstrates that STAT4 single nucleotide polymorphism affects clinical outcomes of pediatric acute leukemia patients after hematopoietic stem cell transplant. PMID: 27960128
- This study shows that STAT4 gene polymorphisms are associated with ankylosing spondylitis in Southwest China. PMID: 27394003
- This study found a significant association between STAT4 rs7582694 alleles and genotypes and susceptibility to endometriosis in a population for the first time. PMID: 27235632
- This study concludes that STAT4-rs7574865 polymorphism is clearly associated with the risk of Rheumatoid Arthritis in the Western Algerian population. PMID: 25351936
- Results demonstrate that STAT4 rs7574865 and IRF5 rs2004640G/T substitution are associated with a susceptibility to systemic sclerosis. PMID: 26712637
- Findings indicate that although STAT4 and IFIH1 SNPs are not associated with Type 1 Diabetes (T1D) in a Brazilian population, they might play a role in susceptibility to T1D on a larger worldwide scale. PMID: 26782418
- SNP rs7574865 in STAT4 might contribute to the progression to Hepatocellular Carcinoma (HCC). PMID: 26745093
- Five SNPs (rs7574865 in STAT4, rs9267673 near C2, rs2647073 and rs3997872 near HLA-DRB1 and rs9275319 near HLA-DQ), were found to be significantly associated with the risk of HBV-related LC. PMID: 26538132
- There is a significant association between STAT4 rs7574865 polymorphism and Inflammatory Bowel Disease (IBD) susceptibility in the overall population. PMID: 26066297
- STAT4 protein genetic variation is a prognostic factor predicting the treatment with interferon-alpha therapy in chronic hepatitis B. PMID: 26704347
- The presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. PMID: 26569609
- STAT4 rs7574865 polymorphism may be associated with a significantly reduced risk of HBV-induced HCC in Asian populations. PMID: 25178516
- Gene polymorphisms along with Ptpn22 polymorphism confer susceptibility to rheumatoid arthritis in all major ethnic groups. [meta-analysis] PMID: 25963842
- Significant association with alleles of two STAT4 markers and nominal association of Autoimmune Addison's disease with alleles at GATA3, is reported. PMID: 24614117
- Our replication study showed that the rs7574865 in STAT4 and rs9275319 in HLA-DQ were not associated with CHB-related HCC in a Korean population. PMID: 25913043
- CCR1, KLRC4, IL12A-AS1, STAT4, and ERAP1 are bona fide susceptibility genes for Behcet's disease. PMID: 26097239
- STAT4 rs7574865 G/T and PTPN22 rs2488457 G/C polymorphisms are identified as susceptibility factors for Juvenile Idiopathic Arthritis (JIA). PMID: 25781893
- The STAT4 minor allele may be associated with the spontaneous clearance of HBV, whereas the major allele may be associated with the progression of HBV-related liver disease. PMID: 25829184
- STAT4 may inhibit HCC development by modulating HCC cell proliferation. PMID: 25852285
- Review/Meta-analysis: STAT4 rs7574865 polymorphism confers susceptibility to rheumatoid arthritis in major ethnic groups. PMID: 24751105
- Evidence of a higher risk of developing pericarditis with STAT4 genotypes, and an association between HCP5 rs3099844 and anti-Ro/SSA antibodies in Italian systemic lupus erythematosus patients. PMID: 25369137
- STAT4 rs7574865 appears to be Tibetan specific in hepatitis B virus natural clearance. PMID: 25041342
- STAT4 rs7574865 did not seem to correlate with hepatitis B virus infection susceptibility or natural clearance; its role in hepatocellular carcinoma development seemed ambiguous. PMID: 25365208
- Loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA. PMID: 25486484
- Increased expression of STAT4 is positively correlated with the depth of invasion in colorectal cancer patients. PMID: 25864744
- This work reported the association of 14q32.11 (EFCAB11) with Hepatocellular carcinoma in the Chinese Han population and revealed the genetic interaction between STAT4 (2q32.2-q32.3) and EFCAB11 (14q32.11) in Hepatocellular carcinoma. PMID: 25665738
- STAT4 rs7574865/rs10181656 polymorphisms increase the risk of autoimmune thyroid diseases in a Chinese population. PMID: 25019342
- Rheumatoid arthritis cases showed a significantly higher frequency of the STAT4 T allele carriage (GT+TT genotypes) compared to controls. PMID: 24979672
- STAT4 plays a crucial role in the function of innate and adaptive immune cells; dysregulated expression and aberrant activation of STAT4 is observed in many human autoimmune conditions. PMID: 24844303
Q&A
What are the primary experimental applications of biotin-conjugated STAT4 antibodies in immunological studies?
Biotin-conjugated STAT4 antibodies are primarily employed for protein localization, phosphorylation status analysis, and cytokine signaling pathway characterization. Key applications include:
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Western blotting: Detect STAT4 expression in thymus, spleen, or testis lysates under IL-12 stimulation .
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Flow cytometry: Identify STAT4+ immune subsets (e.g., Th1 cells, dendritic cells) in PBMCs or tissue homogenates .
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Immunoprecipitation: Study STAT4 homodimer/heterodimer formation with STAT1 or STAT3 .
For IL-12 signaling studies, pre-activate cells with 10–20 ng/mL IL-12 for 15–30 minutes before lysate preparation to induce tyrosine phosphorylation . Always include isotype-matched controls to distinguish nonspecific binding in flow cytometry assays .
How should researchers validate the specificity of STAT4 biotin-conjugated antibodies across experimental models?
Validation requires a three-pronged approach:
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Knockout controls: Use STAT4-deficient murine splenocytes or CRISPR-edited cell lines to confirm absence of signal .
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Cross-reactivity testing: Verify antibody specificity against STAT1 and STAT3 due to 47–52% sequence homology .
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Functional blocking: Pre-incubate antibodies with recombinant STAT4 protein (10 μg/mL, 1 hour) to assess signal reduction.
Source demonstrates that chemotherapy-treated patient samples show reduced STAT4 levels, providing a natural negative control for validation (MFI reduction from 50 ± 4 to 32 ± 5 in lymphocytes).
What are the critical parameters for optimizing STAT4 detection in low-abundance samples?
For flow cytometry, permeabilize cells with 90% methanol for 10 minutes at −20°C to improve intracellular STAT4 detection .
How can researchers resolve discrepancies in STAT4 expression levels observed across cancer models?
Contradictory findings often arise from tumor microenvironment heterogeneity and post-translational modifications. In breast cancer:
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STAT4-high tumors: Correlate with PD-L1 upregulation via IL-12R/JAK2/STAT3 axis, enhancing immunotherapy response .
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STAT4-low tumors: Associate with radiotherapy resistance mediated by MALAT1/miR-21-5p/THRB dysregulation .
Methodological adjustments:
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Stratify analyses by IL-12/IFN-γ secretion levels using ELISA.
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Perform phospho-STAT4 (Tyr693) staining to differentiate active vs. total STAT4 pools .
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Utilize spatial transcriptomics to map STAT4 expression in tumor vs. stromal compartments .
What protocols mitigate STAT4 degradation in clinical samples from chemotherapy-treated patients?
Chemotherapeutic agents like etoposide reduce STAT4 half-life from 8.1 ± 1.2 hrs to 3.4 ± 0.7 hrs . Mitigation strategies include:
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Proteasome inhibition: Add 20 μM MG-132 during lysis to prevent ubiquitin-mediated degradation .
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Rapid processing: Isolate PBMCs within 2 hrs of collection; avoid freeze-thaw cycles.
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Subset enrichment: Use CD56+/CD3+ magnetic beads to concentrate STAT4+ NK/T cells prior to analysis .
For longitudinal studies, collect pre-/post-chemotherapy paired samples and normalize STAT4 levels to β-actin or GAPDH .
How does STAT4’s dual role in pro-inflammatory signaling and tumor suppression influence experimental design?
STAT4 exhibits context-dependent functionality:
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Immunological assays: In DCs/macrophages, IL-12 induces STAT4-dependent IFN-γ production, enhancing microbicidal activity against T. gondii . Use 10 ng/mL IL-12 + 50 ng/mL IL-18 co-stimulation to maximize this effect .
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Oncological assays: In breast cancer, STAT4 overexpression upregulates PD-L1 but requires concurrent JAK2 inhibition to block STAT3-mediated immunosuppression .
Experimental recommendations:
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For immunotherapy studies, calculate the STAT4-related pathway score (Srps) using JAK2, STAT3, STAT4, CD274, IL12RB1/2 expression to predict anti-PD-1 response .
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In radiotherapy resistance models, transfect cells with STAT4-overexpression vectors (MOI 10–20) and quantify MALAT1 via qRT-PCR .
