GATL4 Antibody

What is GATA4 and why is it important in research?

GATA4 is a zinc finger transcription factor that plays critical roles in various developmental and physiological processes. It functions as an essential regulator of metabolic, immune, and microbial regionalization between the proximal and distal small intestine . The protein is also crucial for cardiac development and function. Research utilizing GATA4 antibodies allows scientists to investigate its expression patterns, phosphorylation states, and functional significance in different tissues and disease models. Understanding GATA4's regulatory mechanisms provides insights into developmental biology, cardiovascular diseases, intestinal disorders, and potential therapeutic targets .

What types of GATA4 antibodies are available for research?

Current research employs several types of GATA4 antibodies, each with specific applications:

How do I select the appropriate GATA4 antibody for my research?

When selecting a GATA4 antibody, multiple factors should guide your decision process. First, determine which application(s) you need the antibody for - whether Western blotting, immunohistochemistry, immunofluorescence, ELISA, or flow cytometry. Review the validation data for each antibody in your specific application . Consider the species reactivity - ensure the antibody recognizes GATA4 in your experimental model organism (human, mouse, rat) .
For mechanistic studies, phospho-specific antibodies targeting Ser105 or Ser262 may be more appropriate, as these post-translational modifications regulate GATA4 activity . If you're studying regional expression patterns, particularly in the intestine where GATA4 shows distinct expression profiles, select antibodies validated for immunohistochemistry and with demonstrated specificity in intestinal tissues . For quantitative studies, monoclonal antibodies typically offer greater consistency between experiments and lots.

What are the basic applications of GATA4 antibodies in research?

GATA4 antibodies support multiple research applications:

• Western Blotting (WB): For protein expression quantification and validation of GATA4 presence in tissue/cell lysates. Most GATA4 antibodies (17 out of 19 in the catalog) are validated for WB applications .

• Immunohistochemistry (IHC): For visualizing GATA4 expression patterns in tissue sections, particularly useful for studying regionalization in intestinal tissues. Several antibodies (at least 7) are specifically validated for IHC .

• Immunofluorescence (IF): For co-localization studies with other proteins and precise cellular localization. Multiple antibodies support this application .

• ELISA: For quantitative measurement of GATA4 in solution. Several antibodies are validated for this application .

• Flow Cytometry: For studying GATA4 expression in specific cell populations. Two monoclonal antibodies ([PCRP-GATA4-1A7]) are specifically validated for flow cytometry .

How can I optimize GATA4 antibody performance for detecting regional expression in intestinal tissue?

Optimizing GATA4 antibody performance for intestinal regionalization studies requires careful consideration of several technical factors. First, tissue preservation method significantly impacts epitope accessibility - fresh frozen samples often provide better signal than formalin-fixed tissues for certain antibodies. When using GATA4 antibodies for proximal-distal intestinal regionalization studies, heat-mediated antigen retrieval in citrate buffer (pH 6.0) typically yields superior results .
For immunofluorescence studies examining GATA4's role in controlling bacterial colonization and inflammatory tissue immunity, implement a blocking step with 5-10% normal serum matching the secondary antibody host species, plus 0.1-0.3% Triton X-100 for membrane permeabilization . When studying GATA4's relationship with retinol metabolism and luminal IgA, counterstain with DAPI and use thin sections (5μm) to achieve optimal signal-to-noise ratios. Particularly important for intestinal studies is the use of controls from GATA4-deficient regions (distal small intestine) alongside GATA4-expressing regions (proximal small intestine) to validate antibody specificity in the context of intestinal tissue .

How can phospho-specific GATA4 antibodies be utilized to study signaling mechanisms?

Phospho-specific GATA4 antibodies provide critical insights into signaling pathways regulating GATA4 activity. GATA4 contains multiple phosphorylation sites, with Ser105 and Ser262 being particularly important for its function . To effectively utilize phospho-specific antibodies for signaling studies, researchers should implement the following methodology:

• Baseline vs. Stimulated Conditions: Compare phosphorylation levels under resting conditions versus following stimulation with factors known to activate GATA4 (e.g., hypertrophic stimuli in cardiomyocytes).

• Kinase Inhibitor Studies: Pre-treat samples with specific kinase inhibitors to identify the responsible signaling pathway. For Ser105 phosphorylation, which is often mediated by ERK/MAPK pathways, use MEK inhibitors (PD98059, U0126) as controls.

• Phosphatase Controls: Include phosphatase-treated samples to validate antibody specificity for the phosphorylated epitope.

• Positive Controls: Include samples from systems with known high levels of GATA4 phosphorylation (such as hypertrophic heart tissue).

• Validation Strategy: Confirm phospho-specific antibody results using complementary approaches such as phospho-tag gels or mass spectrometry.
  For studying the relationship between GATA4 phosphorylation and intestinal regionalization functions, combine phospho-specific antibodies with functional readouts such as retinol metabolism markers or IgA production to establish mechanistic connections .

What strategies can be employed to investigate GATA4's role in intestinal regionalization and immunity?

Investigating GATA4's role in intestinal regionalization and immunity requires multifaceted approaches that combine antibody-based techniques with functional assays. Based on recent research, the following strategic framework is recommended:

• Conditional Knockout Models: Utilize intestinal epithelium-specific GATA4 knockout models (GATA4ΔIEC) alongside controls to examine changes in bacterial colonization patterns and immune responses .

• Spatial Mapping: Employ GATA4 antibodies for precise spatial mapping of expression along the intestinal tract, focusing on the proximal-distal axis where GATA4 shows differential expression patterns .

• Multi-parameter Analysis: Combine GATA4 immunostaining with markers for retinol metabolism (CRBP2, RDH10), IgA production, and inflammatory cytokines (particularly IL-17) to establish mechanistic relationships .

• Microbiome Analysis: Correlate GATA4 expression patterns with bacterial colonization, particularly focusing on segmented filamentous bacteria which are influenced by GATA4-regulated mechanisms .

• Cross-species Validation: Extend findings from mouse models to human samples, particularly from patients with conditions like celiac disease where GATA4 expression may be altered .
  The research demonstrates that GATA4 controls bacterial colonization and inflammatory tissue immunity by regulating retinol metabolism and luminal IgA. In models lacking jejunal GATA4 expression, commensal segmented filamentous bacteria promoted pathogenic inflammatory responses, disrupting barrier function and increasing mortality during infection challenges .

How can I troubleshoot non-specific binding when using GATA4 antibodies?

Non-specific binding is a common challenge when working with GATA4 antibodies, particularly in complex tissues like intestine where multiple cell types are present. To troubleshoot this issue:

• Antibody Titration: Systematically test a range of primary antibody dilutions (1:100 to 1:2000) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Blocking Optimization: Extend blocking times (2-3 hours at room temperature) and test different blocking agents (BSA, normal serum, commercial blockers) to reduce non-specific interactions.

• Washing Procedures: Implement more stringent washing protocols, including longer wash times and increased detergent (0.1-0.5% Tween-20) in wash buffers.

• Validation Controls:

  • Use tissues/cells known to be negative for GATA4 (distal small intestine sections)

  • Include peptide competition assays where the antibody is pre-incubated with its target peptide

  • Use siRNA or CRISPR-generated GATA4 knockdown cells as negative controls

• Secondary Antibody Optimization: Test secondary antibodies from different vendors and consider highly cross-adsorbed versions to minimize cross-reactivity.
  For particularly challenging samples, consider signal amplification systems like tyramide signal amplification, but be aware these may increase background if not carefully optimized.

How should I design experiments to study GATA4's role in disease models?

Designing robust experiments to investigate GATA4's role in disease models requires careful planning and appropriate controls. Based on recent research findings, implement the following experimental design framework:

• Model Selection and Validation:

  • For intestinal disorders: Use region-specific sampling (proximal vs. distal intestine) as GATA4 shows distinct regional expression patterns

  • For cardiovascular studies: Consider developmental stage and disease progression when selecting tissue harvest timepoints

  • Include both acute and chronic disease models where appropriate

• Appropriate Controls Structure:

  • Wild-type controls matched for age, sex, and genetic background

  • Heterozygous controls to assess gene dosage effects

  • Region-matched controls (e.g., proximal vs. distal intestine)

  • Treatment-matched controls for intervention studies

• Comprehensive Readouts:

  • GATA4 expression analysis: Protein (various antibodies), mRNA (qPCR), and activity (ChIP)

  • Downstream target assessment: Known GATA4 transcriptional targets

  • Functional outcomes: Tissue-specific functional measures

  • For intestinal studies: Barrier function, bacterial colonization, immune cell profiles

• Temporal Considerations:

  • Developmental timepoints for congenital disorders

  • Disease progression timepoints for acquired conditions

  • Consider circadian variations in GATA4 expression

• Statistical Design:

  • Power analysis to determine appropriate sample sizes

  • Blinded analysis to prevent observer bias

  • Appropriate statistical tests based on data distribution
    When studying GATA4's role in infection responses, include challenge models like Citrobacter rodentium with careful monitoring of barrier function, inflammatory markers, and mortality outcomes .

What are the recommended protocols for dual immunofluorescence using GATA4 antibodies?

Dual immunofluorescence protocols involving GATA4 antibodies require careful optimization to ensure proper co-detection with other markers. Based on research methodologies, the following protocol framework is recommended:

• Sample Preparation:

  • Fresh frozen sections (7-10μm) typically yield better results than FFPE for dual staining

  • For FFPE tissues, extended antigen retrieval (20 minutes in citrate buffer pH 6.0) improves GATA4 detection

• Blocking Strategy:

  • Block with 5-10% normal serum from host species of both secondary antibodies

  • Add 0.3% Triton X-100 for nuclear antigen access

  • Include 1% BSA to reduce non-specific binding

  • Block for extended period (2 hours at room temperature)

• Antibody Selection and Order:

  • When combining rabbit GATA4 antibodies with other rabbit-raised antibodies, use sequential immunostaining with Fab fragment blocking between rounds

  • For co-localization with transcription factors, select GATA4 antibodies validated for nuclear detection

  • When studying intestinal tissue, polyclonal antibodies generally provide better signal in this context

• Optimization Guidelines:

  • Test a range of antibody concentrations (typically 1:200-1:1000 for primary)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use highly cross-adsorbed secondary antibodies to prevent cross-reactivity

  • Include appropriate controls (single-stained, secondary-only, isotype)

• Advanced Considerations:

  • For phospho-GATA4 detection, include phosphatase inhibitors in all buffers

  • When combining with weak signals, apply tyramide signal amplification to the weaker antibody only

  • For intestinal studies, include autofluorescence reduction steps (0.1% Sudan Black in 70% ethanol for 20 minutes)
    This methodology enables successful co-localization of GATA4 with other markers important for understanding its functional relationships in tissue contexts.

How can GATA4 antibodies be applied in ChIP assays to study transcriptional regulation?

Chromatin Immunoprecipitation (ChIP) assays using GATA4 antibodies provide crucial insights into GATA4's direct transcriptional targets. The following methodology optimizes ChIP performance with GATA4 antibodies:

• Antibody Selection for ChIP:

  • Choose antibodies validated specifically for ChIP applications

  • Use monoclonal antibodies for higher consistency and specificity

  • Target antibodies against the DNA-binding domain may have reduced efficiency

  • Select antibodies recognizing epitopes outside regions involved in DNA-protein interactions

• Sample Preparation Optimization:

  • Crosslinking time significantly affects GATA4 ChIP: optimize between 10-15 minutes with 1% formaldehyde

  • Include two-step crosslinking (1% formaldehyde followed by EGS/DSG) for improved GATA4-DNA complex stability

  • Sonication conditions: 10-15 cycles (30s on/30s off) to achieve fragments of 200-500bp

• IP Protocol Considerations:

  • Use 3-5μg of GATA4 antibody per IP reaction

  • Extended incubation times (overnight at 4°C with rotation)

  • Include protease and phosphatase inhibitors in all buffers

  • Add BSA (0.1-0.5%) to reduce non-specific binding

• Controls Structure:

  • Input control (10% of starting chromatin)

  • IgG control matched to GATA4 antibody host species

  • Positive control: IP with antibody against abundant histone mark

  • Positive control target: known GATA4 binding site (ANF promoter for cardiac studies)

  • Negative control region: genomic region without GATA4 binding sites

• Analysis Approach:

  • qPCR for known and predicted binding sites

  • ChIP-seq for genome-wide binding profile

  • Integrate with RNA-seq data to identify functional binding events
    This methodology enables comprehensive analysis of GATA4's direct transcriptional targets, particularly those involved in intestinal regionalization and immunity regulation .

How can researchers resolve discrepancies in GATA4 detection between different antibodies?

Discrepancies between different GATA4 antibodies can arise from several factors and require systematic troubleshooting:

• Epitope Mapping Analysis:

  • Compare the epitope regions recognized by different antibodies

  • Antibodies recognizing N-terminal vs. C-terminal epitopes may yield different results

  • Phospho-specific antibodies (Ser105, Ser262) will only detect modified forms

• Isoform Recognition Profile:

  • GATA4 has multiple isoforms - verify which isoforms your antibodies recognize

  • Review literature to confirm which isoforms are present in your experimental system

  • Consider using isoform-specific primers for RT-PCR validation alongside antibody detection

• Cross-Reactivity Assessment:

  • Test antibodies in GATA4 knockout/knockdown systems

  • Perform Western blots with recombinant GATA4 alongside related family members (GATA1-6)

  • Conduct peptide competition assays to confirm specificity

• Technical Optimization Matrix:

  • Create a matrix testing multiple antibodies across different:

    • Fixation methods (PFA, methanol, acetone)

    • Antigen retrieval approaches (heat, enzymatic, pH variations)

    • Blocking reagents (BSA, normal serum, commercial blockers)

    • Incubation conditions (time, temperature, concentration)

• Validation Strategy:

  • Correlate protein detection with mRNA expression

  • Compare results with published expression patterns

  • For intestinal studies, utilize the known proximal-distal gradient as internal validation
    When antibodies show discrepancies, prioritize results from monoclonal antibodies and those with extensive validation in your specific application and tissue type.

What are the best practices for quantifying GATA4 expression in tissue samples?

Quantifying GATA4 expression accurately requires adherence to methodological best practices tailored to the specific application:

• Western Blot Quantification:

  • Use recombinant GATA4 standards to create a calibration curve

  • Load equal total protein (validated by housekeeping proteins or total protein stains)

  • Image using a linear detection system (avoid film exposure)

  • Normalize to appropriate loading controls (nuclear proteins for nuclear GATA4)

  • Include positive controls with known GATA4 expression levels

• Immunohistochemistry/Immunofluorescence Quantification:

  • Use automated image analysis software with validated algorithms

  • Standardize acquisition parameters (exposure, gain, offset)

  • Analyze nuclear intensity within defined cell populations

  • Count percentage of GATA4-positive cells in defined regions

  • For intestinal studies, quantify expression along the proximal-distal axis

• Flow Cytometry Approach:

  • Use antibodies specifically validated for flow cytometry

  • Include FMO (Fluorescence Minus One) controls

  • Use appropriate permeabilization for nuclear GATA4 detection

  • Report data as mean fluorescence intensity (MFI) and percent positive

• RT-qPCR Complementary Analysis:

  • Use validated GATA4-specific primers

  • Include multiple reference genes for normalization

  • Correlate mRNA with protein levels to identify post-transcriptional regulation

• Statistical Considerations:

  • Use appropriate statistical tests based on data distribution

  • Report both biological and technical replicates

  • Consider power analysis to determine sample size

  • For region-specific analysis, use matched samples from different intestinal regions
    Following these quantification practices ensures reliable measurement of GATA4 expression differences in experimental systems.

How can researchers integrate GATA4 antibody data with functional genomics approaches?

Integrating GATA4 antibody data with functional genomics requires coordinated multi-omics strategies:

• ChIP-seq and Antibody-based Detection Integration:

  • Use GATA4 antibodies for ChIP-seq to identify genome-wide binding sites

  • Correlate binding patterns with region-specific expression detected by immunohistochemistry

  • Validate key binding sites with targeted ChIP-qPCR

  • Connect binding data with expression changes in GATA4 knockout/knockdown models

• Multi-omics Integration Framework:

  • Combine GATA4 protein data (antibody-based) with:

    • Transcriptomics (RNA-seq) to identify direct and indirect targets

    • Epigenomics (ATAC-seq, histone ChIP-seq) to understand chromatin context

    • Proteomics to identify GATA4 interaction partners

    • Metabolomics to connect with functional outcomes (e.g., retinol metabolism)

• Spatial Transcriptomics Correlation:

  • Correlate GATA4 antibody staining patterns with spatial transcriptomics data

  • Focus on regional differences in intestinal expression patterns

  • Create integrated spatial maps of GATA4 activity

• Network Analysis Approach:

  • Use GATA4 antibody data as validation for predicted regulatory networks

  • Construct tissue-specific gene regulatory networks centered on GATA4

  • Validate key network connections experimentally using genetic perturbations

• Translational Integration:

  • Connect mouse model findings with human patient samples

  • Correlate GATA4 expression patterns with disease biomarkers

  • For intestinal studies, integrate with microbiome data given GATA4's role in regulating bacterial colonization
    This integrated approach provides a comprehensive understanding of GATA4's functional role in various biological contexts, particularly in intestinal regionalization and immunity regulation .