GATA6 Monoclonal Antibody

What is GATA6 and what are its primary biological functions?

GATA6 is a zinc finger transcription factor that plays crucial roles in regulating terminal differentiation and proliferation in various tissues. It functions as a transcriptional activator that regulates SEMA3C and PLXNA2 gene expression . In cardiac development, GATA6 directs human precardiac mesoderm patterning during early cardiogenesis . GATA6 is also involved in gene regulation specifically in the gastric epithelium and may regulate genes that protect epithelial cells from bacterial infection .

In human skin, GATA6 controls several physiological processes contributing to homeostasis of the upper pilosebaceous unit by triggering ductal and sebaceous differentiation while limiting cell proliferation and lipid production to prevent hyperseborrhoea . Additionally, GATA6 mediates the effects of retinoic acid on sebocyte proliferation and contributes to immune regulation by modulating the expression of inflammatory genes .

What is the molecular weight of GATA6 protein and how is it detected in Western blot applications?

GATA6 has a molecular weight of approximately 55-60 kDa as detected in Western blot applications. According to validation studies, GATA6 appears as a specific band at approximately 55 kDa when using antibodies such as the Goat Anti-Human GATA-6 Antigen Affinity-purified Polyclonal Antibody . The Cell Signaling Technology's GATA-6 (D61E4) XP® Rabbit mAb also detects endogenous GATA6 at 55 kDa .

For optimal Western blot detection, researchers typically use:

• 1 μg/mL concentration for polyclonal antibodies

• 1:1000 dilution for monoclonal antibodies

• Reducing conditions and appropriate immunoblot buffer systems

Which tissues and cell lines express GATA6 at detectable levels?

GATA6 expression has been validated in multiple cell lines and tissues:

GATA6 staining is typically localized to nuclei, which is consistent with its function as a transcription factor . In human embryonic stem cells (hESCs) undergoing cardiac differentiation, GATA6 expression is dynamically regulated and plays crucial roles in early mesoderm patterning stages .

What are the key differences between monoclonal and polyclonal GATA6 antibodies for research applications?

Monoclonal GATA6 Antibodies:

• Recognize a single epitope on the GATA6 protein

• Examples include Mouse Anti-Human GATA-6 (Clone #222228, MAB1700) and GATA-6 (D61E4) XP® Rabbit mAb

• Offer high specificity and reproducibility between lots

• Often optimized for specific applications (e.g., Clone #222228 is validated for ICC/IF at 10-15 μg/mL)

• Typically produced by immunizing animals with synthetic peptides (e.g., peptides near the amino terminus of human GATA-6)

Polyclonal GATA6 Antibodies:

• Recognize multiple epitopes on the GATA6 protein

• Examples include Goat Anti-Human GATA-6 Antigen Affinity-purified Polyclonal Antibody (AF1700)

• Often generated against recombinant proteins (e.g., E. coli-derived recombinant human GATA-6, Met1-Thr449)

• May provide stronger signals due to binding to multiple epitopes

• Typically validated for broader application ranges (e.g., WB, ICC/IF, IHC, ChIP)

Selection should be based on experimental needs, with monoclonal antibodies preferred for high-specificity applications and polyclonal antibodies for detecting low abundance targets or for applications like ChIP.

How should researchers validate GATA6 antibody specificity for their experimental systems?

Researchers should implement a comprehensive validation strategy:

• Positive and negative controls:

  • Use cell lines with known GATA6 expression levels (e.g., HCT-116, KATO-III)

  • Include GATA6 knockout/knockdown samples as negative controls

  • For ChIP experiments, use IgG control antibodies (e.g., AB-108-C)

• Multiple detection methods:

  • Confirm antibody performance across multiple applications (WB, IHC, IF)

  • Compare results between different antibody clones/sources

  • Verify expected subcellular localization (nuclear for GATA6)

• Blocking peptide competition:

  • Perform assays with and without specific blocking peptides

  • Observe signal reduction in presence of blocking peptide

• Cross-reactivity assessment:

  • Test antibody against closely related proteins (other GATA family members)

  • Verify species cross-reactivity when working with non-human samples

• Literature comparison:

  • Compare results with published GATA6 expression patterns

  • Reference antibody validation studies such as those conducted by manufacturers

Which GATA6 antibody clones have the most extensive validation for research applications?

Several GATA6 antibody clones have undergone extensive validation:

• Clone #222228 (Mouse Anti-Human GATA-6 Monoclonal, MAB1700):

  • Validated for ICC in HCT-116 human colorectal carcinoma and HT-29 human colon adenocarcinoma cell lines

  • Specific nuclear staining confirmed with appropriate counterstaining

  • Recommended concentration: 10-15 μg/mL for ICC applications

• D61E4 XP® Rabbit mAb (Cell Signaling Technology, #5851):

  • Validated for Western blotting, IHC, IF, Flow cytometry, ChIP, CUT&RUN, and CUT&Tag

  • Recognizes endogenous levels of total GATA-6 protein

  • Species reactivity confirmed for human and mouse samples

  • Cited in 76 publications according to supporting data

• Polyclonal Goat Anti-Human GATA-6 (AF1700):

  • Validated for Western blot, ICC/IF, IHC, ChIP, and Simple Western applications

  • Demonstrated specificity in detecting GATA-6 in multiple cell lines including KATO-III and PC-3

  • Successfully used to detect GATA-6-regulated genes by ChIP

These antibodies have established reproducibility records and extensive application validation, making them suitable for various research contexts.

What are the optimal conditions for using GATA6 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For optimal GATA6 ChIP experiments, the following protocol elements have been validated:

Sample Preparation:

• Fix cells using formaldehyde to crosslink protein-DNA complexes

• Resuspend in appropriate lysis buffer

• Sonicate to shear chromatin to appropriate fragment sizes (200-500 bp ideal)

Immunoprecipitation Conditions:

• Use 5-10 μg of GATA6 antibody per ChIP reaction

• For D61E4 XP® Rabbit mAb: Use 10 μl of antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per IP

• For Goat Anti-Human GATA-6 (AF1700): Use 5 μg with 15 minutes incubation in an ultrasonic bath

• Include appropriate negative control antibody (e.g., normal IgG)

Detection Methods:

• Standard PCR for specific target regions (e.g., mucin4 promoter has been validated)

• qPCR for quantitative analysis

• ChIP-seq for genome-wide binding profile analysis

Validated Cell Lines:

• KATO-III human gastric carcinoma cells have been successfully used for GATA6 ChIP experiments

• hESCs undergoing cardiac differentiation have shown successful GATA6 binding detection through CUT&RUN analysis

Research has shown GATA6 binding to WNT and BMP pathway genes during early mesoderm patterning, with significant binding peaks detected near cardiac development genes .

How should researchers optimize immunofluorescence protocols for detecting GATA6 in different cell types?

Protocol Optimization for GATA6 Immunofluorescence:

• Fixation and Permeabilization:

  • Use immersion fixation for cultured cells (as validated in HCT-116, HT-29, and KATO-III cells)

  • Test different fixatives (4% paraformaldehyde is commonly used)

  • Ensure adequate permeabilization to access nuclear GATA6

• Antibody Concentrations and Incubation:

  • For monoclonal antibodies (e.g., MAB1700): Use 10-15 μg/mL concentration

  • For D61E4 XP® Rabbit mAb: Use 1:800-1:1600 dilution

  • For polyclonal antibodies (e.g., AF1700): Use 10 μg/mL concentration

  • Optimize incubation time (3 hours at room temperature has been validated)

• Secondary Antibody Selection:

  • Use fluorescently conjugated secondary antibodies matching the host species

  • Examples include NorthernLights™ 557-conjugated Anti-Mouse IgG (NL007) or Anti-Goat IgG (NL001)

  • Include DAPI counterstain to visualize nuclei and confirm nuclear localization

• Controls and Validation:

  • Include negative controls (primary antibody omission, isotype controls)

  • Use cell lines with known GATA6 expression patterns

  • Confirm nuclear localization pattern consistent with transcription factor function

• Signal Amplification (if needed):

  • Consider tyramide signal amplification for low abundance

  • Use confocal microscopy for improved signal-to-noise ratio and localization accuracy

Specific staining of GATA6 should be localized to nuclei when properly optimized, as observed in multiple validated cell lines .

What is the importance of GATA6 in cardiac development research and how can antibodies help elucidate its function?

GATA6 plays critical roles in cardiac development, with several key research areas benefiting from antibody-based investigations:

GATA6 in Cardiac Lineage Specification:

• GATA6 loss-of-function in human embryonic stem cells (hESCs) causes profound impairment in cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation

• Antibodies enable tracking of GATA6 expression dynamics during different stages of cardiac differentiation

• Flow cytometry using GATA6 antibodies allows quantification of cardiac progenitor populations

Regulation of Key Developmental Pathways:

• GATA6 regulates WNT and BMP programs essential for early cardiac development

• ChIP and CUT&RUN experiments using GATA6 antibodies have identified direct target genes including those in WNT and BMP networks

• Research using GATA6 antibodies has revealed that GATA6 interacts with important developmental transcription factors and chromatin remodelers during early mesoderm patterning

Clinical Relevance:

• GATA6 haploinsufficiency is associated with congenital heart disease (CHD) with variable comorbidity

• Antibody-based studies help understand how GATA6 mutations impact cardiac development

• Immunohistochemistry with GATA6 antibodies enables assessment of expression patterns in patient-derived tissues

Experimental Approach Using Antibodies:

• Western blotting to monitor GATA6 protein levels during differentiation

• Flow cytometry to quantify cardiac progenitor markers (e.g., KDR, PDGFRα) in GATA6 mutant cells

• ChIP and CUT&RUN experiments to identify GATA6 binding sites in cardiac regulatory regions

• Immunoprecipitation to identify GATA6 protein interaction partners (e.g., EOMES, SMARCC1)

Research has shown that modulating WNT and BMP inputs during the first 48 hours of cardiac differentiation is sufficient to partially rescue CPC and CM defects in GATA6 heterozygous and homozygous mutant hESCs .

How can GATA6 antibodies be used in CUT&RUN and CUT&Tag experiments to profile chromatin binding?

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and CUT&Tag (Cleavage Under Targets and Tagmentation) represent advanced techniques for profiling transcription factor binding with higher signal-to-noise ratios than traditional ChIP-seq. For GATA6:

Optimized CUT&RUN Protocol:

• The D61E4 XP® Rabbit mAb has been validated for CUT&RUN at 1:50 dilution using CUT&RUN Assay Kit #86652

• Antibody specificity is critical as these techniques amplify specific binding signals

• GATA6 CUT&RUN analysis during early mesoderm patterning has revealed:

  • Distribution of significant GATA6 binding peaks across the genome

  • Transcription factor motif enrichment at GATA6-bound loci

  • Association with genes related to cardiac development and morphogenesis

CUT&Tag Applications:

• The D61E4 XP® Rabbit mAb has been validated for CUT&Tag at 1:50 dilution using CUT&Tag Assay Kit #77552

• This approach can map GATA6 binding sites with cellular resolution

• Particularly useful for samples with limited material

Data Analysis Insights:

• GATA6 binding has been associated with distal enhancer-like signatures (dELS)

• Integration of GATA6 CUT&RUN data with RNA-seq from GATA6 mutant cells has identified direct regulatory targets

• Analysis reveals GATA6 binding to WNT and BMP pathway genes that are downregulated in GATA6 mutant cells

Genomic Distribution of GATA6 Binding:
CUT&RUN analysis has shown that GATA6 binds to various genomic regions, with significance for genes involved in cardiac development. The binding pattern analysis reveals GATA6's role in regulating key developmental pathways through direct binding to enhancers and promoters .

What protein interactions does GATA6 form during development, and how can they be studied?

GATA6 forms important protein interactions that regulate developmental processes, particularly during cardiac development. These interactions can be studied using various antibody-based approaches:

Identified GATA6 Protein Interactions:

• EOMES: GATA6 interacts with EOMES during early differentiation stages

• SMARCC1: A component of the SWI/SNF chromatin remodeling complex that interacts with GATA6

• SMAD proteins: GATA6 has been found to interact with SMAD2/3 in regulatory complexes

• β-catenin: GATA6 regulates WNT signaling, suggesting potential interactions with β-catenin pathway components

Methodologies for Studying GATA6 Interactions:

• RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins):

  • RIME analysis performed on GATA6-expressing hESCs at day 2 or 4 of cardiac differentiation identified multiple protein interactions

  • The analysis revealed enriched proteins in GATA6 immunoprecipitates compared to controls

• Co-immunoprecipitation (Co-IP):

  • Western blots for GATA6, EOMES, and SMARCC1 performed on GATA6-immunoprecipitated protein lysates confirm interactions

  • These interactions were observed in wild-type samples but absent in GATA6-null samples

• Proximity Ligation Assay (PLA):

  • Can be used to visualize and quantify protein-protein interactions in situ

  • Provides spatial information about where GATA6 interactions occur within cells

• ChIP-re-ChIP:

  • Sequential ChIP using antibodies against GATA6 and its interaction partners

  • Identifies genomic loci co-bound by GATA6 and other factors (e.g., EOMES, SMAD2/3)

  • Research has shown overlap between GATA6 binding sites and those of interaction partners

These interactions suggest that GATA6 functions within multiprotein complexes to regulate gene expression during development, particularly in mesoderm patterning and cardiac specification.

How do GATA6 levels correlate with epithelial differentiation and regeneration in different tissues?

GATA6 plays crucial roles in regulating epithelial differentiation and regeneration across multiple tissue types:

Lung Epithelium:

• Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung

• Loss of Gata6 leads to precocious appearance of BASCs and concurrent loss in epithelial differentiation

• This expansion of BASCs results from increased canonical Wnt signaling in lung epithelium upon Gata6 loss

• Postnatal deletion of Gata6 results in compromised airway regeneration with defective BASC expansion and differentiation

Skin and Pilosebaceous Unit:

• GATA6 is expressed in the upper pilosebaceous unit of normal human skin

• GATA6 is downregulated in acne samples according to microarray datasets

• GATA6 expression in keratinocytes significantly decreases growth rate compared to controls

• GATA6 induction reduces sebocyte proliferation and decreases colony size in clonogenicity assays

Cardiac Tissue:

• GATA6 is required for proper differentiation of cardiac progenitor cells from mesoderm

• GATA6 haploinsufficiency disrupts normal cardiac development, leading to congenital heart defects

• GATA6 regulates a network of genes essential for cardiomyocyte differentiation and function

Regulatory Mechanism:

• GATA6 directly regulates Fzd2, a non-canonical Wnt receptor that inhibits canonical Wnt signaling

• Loss of GATA6 leads to decreased Fzd2 expression and increased canonical Wnt signaling

• This GATA6-Wnt pathway controls the balance between progenitor/stem cell expansion and epithelial differentiation

• Re-expression of Fzd2 or decreased β-catenin expression can rescue, in part, the epithelial defects in Gata6 mutants

These findings demonstrate that GATA6 levels must be precisely regulated to maintain proper balance between stem/progenitor cell expansion and differentiation across multiple epithelial tissues.

What are common issues when using GATA6 antibodies, and how can they be addressed?

Common Issues and Solutions:

• High Background in Immunostaining:

  • Problem: Non-specific staining observed in immunofluorescence or IHC

  • Solution: Optimize blocking (use 5-10% serum matched to secondary antibody host), reduce primary antibody concentration, increase washing steps, and ensure proper permeabilization for nuclear staining

  • Note: Some GATA6 antibodies (e.g., D61E4 XP® Rabbit mAb) show non-specific cytoplasmic staining in limited immune cells in mouse tissues

• Weak or No Signal in Western Blot:

  • Problem: Inability to detect GATA6 band at expected size (~55-60 kDa)

  • Solution: Ensure adequate protein loading (GATA6 is a transcription factor with potentially low expression), optimize antibody concentration (1 μg/mL for polyclonal, 1:1000 for monoclonal antibodies), use enhanced chemiluminescence detection, and verify sample preparation (nuclear extracts may be necessary)

• Multiple Bands in Western Blot:

  • Problem: Detection of multiple bands besides the expected 55-60 kDa band

  • Solution: Use reducing conditions and appropriate immunoblot buffer systems (e.g., Immunoblot Buffer Group 1 has been validated) , consider antibody specificity (polyclonal antibodies may detect additional isoforms), and include appropriate positive controls

• Poor ChIP Efficiency:

  • Problem: Low enrichment in ChIP experiments

  • Solution: Optimize crosslinking conditions, ensure adequate sonication (200-500 bp fragments), increase antibody amount (5-10 μg per reaction), and validate using known GATA6 target genes (e.g., mucin4 promoter)

• Inconsistent Results Between Experiments:

  • Problem: Variability in GATA6 detection across experiments

  • Solution: Standardize protocols (fixation times, antibody concentrations), use consistent lot numbers of antibodies, maintain consistent cell culture conditions as GATA6 expression may be affected by differentiation state, and include standard positive controls

How can researchers integrate GATA6 antibody-based studies with genomic and transcriptomic analyses?

Integrating antibody-based studies with genomic and transcriptomic approaches provides comprehensive insights into GATA6 function:

Integration Strategies:

• ChIP-seq/CUT&RUN with RNA-seq:

  • Combine GATA6 binding profiles with transcriptomic changes in wild-type vs. GATA6 mutant cells

  • This approach has identified direct GATA6 targets during early mesoderm patterning

  • Studies have revealed overlap between GATA6-bound genes and differentially expressed genes in GATA6 mutants

  • Example findings: GATA6 binding peaks associated with decreased differentially expressed genes involved in cardiac development

• Multiomics Analysis:

  • Integrate GATA6 ChIP-seq/CUT&RUN, ATAC-seq, and RNA-seq data

  • This approach has identified GATA6's role in regulating chromatin accessibility

  • GATA6 has been found to interact with chromatin remodelers such as SMARCC1, suggesting cooperative regulation of cardiac lineage gene accessibility

• Motif Analysis and Factor Co-binding:

  • Analyze transcription factor motifs enriched at GATA6 binding sites

  • Integrate with ChIP-seq data for potential co-factors (e.g., EOMES)

  • Approaches like GSEA using datasets of GATA6, EOMES, and SMAD2/3 co-binding have revealed significant overlap in targets

• Visualization and Analysis Tools:

  • Human genome browser representations of GATA6 CUT&RUN data aligned to genes

  • Integration with ENCODE Project data for enhancer-like signatures

  • Heatmaps for visualizing gene expression changes across genotypes (e.g., GATA6+/+, GATA6+/-, GATA6-/-)

Example of Integrated Analysis:
When integrating GATA6 CUT&RUN with RNA-seq data, researchers identified significant GATA6 binding peaks associated with WNT and BMP signaling pathway genes that were downregulated in GATA6 mutant cells. This approach revealed that GATA6 likely directly regulates these pathways through binding to associated enhancers .

What are the implications of GATA6 research for understanding congenital heart disease and potential therapeutic approaches?

GATA6 research using antibody-based approaches has significant implications for understanding congenital heart disease (CHD) mechanisms and developing potential therapies:

Pathophysiological Insights:

• Haploinsufficiency Mechanisms:

  • GATA6 haploinsufficiency is associated with CHD with variable comorbidity of pancreatic or diaphragm defects

  • Research using GATA6+/- hESCs has shown impaired cardiac differentiation, revealing dosage sensitivity

  • GATA6 heterozygous cells showed partially reduced expression of lateral and precardiac mesoderm genes relative to wild-type cells

  • These findings help explain why GATA6 mutations can lead to heart defects even when one allele remains functional

• Early Developmental Origins:

  • GATA6 loss-of-function affects the earliest stages of cardiac differentiation:

    • Mesendoderm and lateral mesoderm patterning

    • Precardiac mesoderm specification

    • Cardiac progenitor cell development

  • This explains the spectrum of cardiac defects observed in patients with GATA6 mutations

• Multi-organ Involvement:

  • GATA6 mutations often cause pancreatic defects alongside CHD

  • Research has demonstrated GATA6's role in both cardiac and definitive endoderm differentiation

  • This explains the co-occurrence of cardiac and other organ defects caused by human GATA6 mutations

Therapeutic Implications:

• Pathway Modulation:

  • Experimental modulation of WNT and BMP inputs during early cardiac differentiation has been shown to partially rescue cardiac defects in GATA6 mutant hESCs

  • This suggests potential therapeutic approaches targeting these pathways in GATA6-related CHD

• Gene Dosage Considerations:

  • GATA6 heterozygous cells show intermediate phenotypes compared to wild-type and homozygous mutants

  • This suggests that even partial restoration of GATA6 function or downstream pathways could provide therapeutic benefit

• Patient-Specific Modeling:

  • GATA6 antibodies enable characterization of patient-derived iPSCs for personalized disease modeling

  • This approach could help predict individual responses to potential therapeutic interventions

• Target Gene Regulation:

  • Identification of direct GATA6 target genes provides potential therapeutic targets

  • Genes in the WNT and BMP pathways that are directly regulated by GATA6 may represent intervention points