GATA4 Antibody
Description
Applications of GATA4 Antibodies
GATA4 antibodies are validated for diverse techniques, including Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry (FACS). Below is a comparative analysis of commercially available antibodies:
Key Notes:
-
PA1-102 and ab227512 are polyclonal, offering broader epitope recognition, while 68012-1-Ig and eBioEvan are monoclonal, ensuring higher specificity .
-
IHC applications often require antigen retrieval (e.g., low pH for eBioEvan) .
Research Findings and Functional Insights
GATA4 antibodies have enabled critical discoveries in developmental biology, cancer, and immune regulation:
Role in Tissue Development and Disease
-
Liver Fibrosis Regression: GATA4 depletion in hepatic stellate cells reduces fibrosis markers (e.g., α-SMA), suggesting therapeutic potential for liver diseases .
-
Heart Development: GATA4 interacts with NKX2.5 and ETS1 to regulate cardiomyocyte and endocardial gene expression, with mutations linked to congenital heart defects .
-
Intestinal Immunity: GATA4 regulates regionalization of bacterial colonization and TH17/IFNγ responses, impacting celiac disease and immunopathology .
Oncogenic and Tumor-Suppressive Roles
-
Breast Cancer Metastasis: GATA4 suppresses MMP9 transcription by inhibiting NF-κB/p65 activity, reducing tumor invasion .
-
Tumor Microenvironment: GATA4 deficiency correlates with reduced lymphocyte infiltration and increased tumor growth in murine models, mediated by CCL2 and CD8+ T-cell activity .
Epigenetic and Transcriptional Regulation
-
Pioneer Activity: GATA4 opens chromatin in collaboration with NKX2.5 and ETS1, creating lineage-specific enhancers in cardiomyocytes and endothelial cells .
-
Senescence and Stress Response: GATA4 induces pro-inflammatory secretory programs linked to senescence, a tumor suppressive mechanism .
Antibody Specificity and Validation
Validation data highlight the importance of epitope targeting and cross-reactivity:
| Antibody | Epitope | Validation Method | Cross-Reactivity |
|---|---|---|---|
| ab227512 | N/A | WB (HeLa, A549, NIH/3T3 lysates) | Human, Mouse, Rat |
| 68012-1-Ig | Full-length protein | WB (PC-3, HEK-293, HepG2 cells) | Human, Mouse |
| PA1-102 | N/A | IHC (human iPSC-derived endoderm) | Human, Mouse |
Critical Considerations:
-
False Positives: Use shRNA knockdown controls (e.g., HeLa cells with GATA4 shRNA) .
-
Optimal Dilution: Varies by application; e.g., PA1-102 requires 1:1000–1:8000 for WB .
Future Directions and Clinical Relevance
Product Specs
Target Background
Q&A
How do researchers validate GATA4 antibody specificity in cardiac differentiation studies?
To ensure antibody specificity:
-
Knockout controls: Use GATA4-deficient iPSCs or CRISPR-edited cell lines as negative controls .
-
Orthogonal validation: Combine western blot (expected band: 50 kDa ) with immunofluorescence (nuclear localization ) and qPCR for GATA4 target genes (e.g., TBX2, PRDM1 ).
-
Cross-reactivity checks: Test against other GATA family members (GATA6 shows 65% homology ) using protein lysates from tissues with differential GATA expression.
What experimental factors cause variability in GATA4 ChIP-seq results across studies?
Key variables include:
BioChIP-seq identifies 15,000+ GATA4 binding sites in adult heart, vs. 1,756 in traditional ChIP , highlighting methodology-dependent outcomes.
How to resolve contradictory data on GATA4’s role in non-cardiac systems?
Discrepancies arise from:
-
Tissue-specific isoforms: Intestinal GATA4 regulates Saa1/2 (pro-TH17 ), while cardiac GATA4 controls TBX2 .
-
Post-translational modifications: Phosphorylation alters DNA-binding affinity in cancer vs. normal cells .
-
Model systems: Mouse intestinal studies show GATA4 loss increases Actinobacillus colonization , whereas iPSC models focus on cardiomyogenesis .
Solution: Contextualize findings using tissue-specific KO models and phospho-specific antibodies .
What advanced applications exist for conjugated GATA4 antibodies?
Conjugation expands functionality:
Why do GATA4 antibodies show differential performance in cancer vs. developmental studies?
-
Epitope masking: Tumorigenic cells overexpress Bmi-1 (a GATA4 pathway gene ), potentially altering antibody accessibility.
-
Clonal variation: sc-25310 detects NTG tumor cells , while eBioEvan exhibits cardiac specificity .
Method: Validate in patient-derived xenografts using dual RNA-FISH (GATA4 mRNA) and IHC .
How to optimize GATA4 antibody dilution for multiplex assays?
Follow iterative testing:
-
Primary antibody titration: Start at 2 µg/mL (WB ), adjust based on background (e.g., 1:200 for IF ).
-
Secondary antibody matching: Use species-specific Fab fragments to prevent cross-reactivity .
-
Signal normalization: Include housekeeping proteins (e.g., Lamin B1 for nuclear quantification ).
What emerging roles of GATA4 require antibody-based investigation?
-
Metastatic niches: GATA4+ tumor cells show enhanced chemoresistance via Bmi-1 upregulation .
-
Immune modulation: Intestinal GATA4 loss correlates with IL-17-driven immunopathology (celiac disease ).
-
Chromatin remodeling: GATA4 recruits histone acetyltransferases to drive H3K27ac deposition at cardiac enhancers .
