GATA6 Antibody

Basic Research Questions

How to validate GATA6 antibody specificity for chromatin immunoprecipitation (ChIP) versus Western blot (WB)?

• Methodological approach:

  • For WB: Use cell lysates with known GATA6 expression (e.g., A549 vs. NCI-H226 lung cancer lines ) and confirm a single band at 60-64 kDa . Include controls with siRNA-mediated GATA6 knockdown (validated by qRT-PCR) to verify target specificity .

  • For ChIP: Perform parallel experiments with IgG isotype controls and validate enriched DNA regions via qPCR or sequencing. Use positive control primers targeting known GATA6-binding promoters (e.g., SEMA3C or PLXNA2) .

What sample preparation protocols optimize GATA6 detection in formalin-fixed paraffin-embedded (FFPE) tissues?

• Key steps:

  • Antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) with microwave treatment (8-15 min) .

  • Block endogenous peroxidases with 3% H₂O₂-methanol (15 min) .

  • Use 3% BSA-PBS for blocking to reduce non-specific binding .

  • Validate with tissues showing differential GATA6 expression (e.g., high in gastric epithelium vs. low in lung adenocarcinoma) .

How to distinguish between polyclonal and monoclonal antibodies for lineage-specific studies?

• Decision matrix:

Advanced Research Questions

How to resolve contradictory reports of GATA6’s oncogenic vs. tumor-suppressive roles?

• Analytical framework:

  • Context-dependent validation:

    • In lung cancer: Use patient-derived xenografts (PDX) to compare GATA6-high (para-tumoral) vs. GATA6-low (tumoral) tissues .

    • In esophageal adenocarcinoma (EAC): Perform siRNA knockdown in OE33 cells to assess apoptosis via BrdU/TUNEL assays .

  • Pathway crosstalk: Profile downstream effectors (e.g., p53/p21 for senescence vs. AKT for survival) .

What methods confirm GATA6’s role in transcriptional regulation during EMT?

• Integrated workflow:

  • Co-stain with EMT markers (E-cadherin, vimentin) in FFPE sections using multiplex IHC .

  • Perform RNA-seq on GATA6-overexpressing A549 cells (DOX-inducible system) to identify regulated genes (e.g., TGFB1, IL6) .

  • Validate binding via CUT&RUN with GATA6 antibody (e.g., 55435-1-AP ) and compare to ATAC-seq data.

How to design studies addressing GATA6’s dual role in differentiation and proliferation?

• Experimental design:

Data Contradiction Analysis

Interpreting conflicting results in GATA6-mediated drug resistance

• Case example:

  • Autophagy induction (NSCLC ) vs. apoptosis suppression (EAC ).

  • Resolution strategy:

    • Compare baseline p53 status (wild-type vs. mutant) across models.

    • Use phospho-specific antibodies (e.g., p-AKT Ser473) to quantify pathway activity .

    • Perform rescue experiments with constitutively active AKT in GATA6-overexpressing cells .

Validating GATA6 as a circulating biomarker in liquid biopsies

• Technical considerations:

  • Use ELISA with antibodies targeting specific epitopes (e.g., C-terminal domain: aa 550–595 ).

  • Cross-validate with mass spectrometry in plasma samples from metastatic vs. localized cancer cohorts .

  • Account for isoform variability by designing primers/probes spanning exon 4–5 junctions .