FOXA3 Antibody, HRP conjugated

What is FOXA3 and what biological roles does it play?

FOXA3 (Forkhead box protein A3), also known as Hepatocyte nuclear factor 3-gamma (HNF-3G) or Transcription factor 3G (TCF-3G), is a transcription factor that acts as a 'pioneer' factor. It opens compacted chromatin for other proteins through interactions with nucleosomal core histones, thereby replacing linker histones at target enhancer and/or promoter sites .

FOXA3 serves multiple critical functions:

• Activates transcription for several liver genes including AFP, albumin, tyrosine aminotransferase, and PEPCK

• Regulates glucose homeostasis by binding to and activating transcription from the G6PC promoter

• Binds to the CYP3A4 promoter and activates its transcription in cooperation with CEBPA

• Interacts with the CYP3A7 promoter alongside members of the CTF/NF-I family

• Participates in neuronal-specific transcription regulation

• May play a role in spermatogenesis regulation

Recent research has also implicated FOXA3 in the development of hepatoblastoma (HB) through upregulation of AFP and HNF1A/MYC expression, while downregulating ZFHX3 expression .

What are the key applications for FOXA3 Antibody, HRP conjugated?

FOXA3 Antibody, HRP conjugated is primarily optimized for the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The HRP conjugation allows direct detection without secondary antibodies, making it suitable for quantitative measurement of FOXA3 expression levels .

While non-HRP conjugated FOXA3 antibodies can be used for additional applications, with appropriate secondary antibody selection:

• Western Blot: Detecting FOXA3 protein expression (approximately 40 kDa observed molecular weight) in cell or tissue lysates .

• Immunohistochemistry (IHC): Localizing FOXA3 in paraffin-embedded tissue sections, particularly in liver, pancreatic, and colon tissues .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Examining subcellular localization of FOXA3 in cultured cells .

When selecting application-specific protocols, researchers should consider the antibody's validation data and optimize conditions for their specific experimental system.

What are the recommended storage and handling conditions for FOXA3 Antibody, HRP conjugated?

For optimal performance and longevity of FOXA3 Antibody, HRP conjugated:

• Storage: Store at -20°C in a non-frost-free freezer

• Buffer composition: Typically supplied in 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles

• Working dilutions: Prepare fresh working dilutions on the day of use

• Light sensitivity: As an HRP-conjugated antibody, protect from prolonged exposure to light

• Stability: When properly stored, remains stable for at least 12 months from date of receipt

• Reconstitution (if lyophilized): Add appropriate volume of distilled water as specified in product documentation

Improper storage or handling can compromise antibody activity and specificity, leading to experimental variability or failure.

What is the molecular weight of FOXA3 and how does this impact detection methods?

FOXA3 has a calculated molecular weight of 37,140 Da, but the observed molecular weight in Western blot applications is approximately 40 kDa . This discrepancy is typical of many transcription factors and may result from:

• Post-translational modifications (phosphorylation, acetylation, etc.)

• The presence of charged amino acid residues affecting migration

• Protein folding effects on electrophoretic mobility

When using FOXA3 Antibody, HRP conjugated for Western blot:

• Expect to observe a band at ~40 kDa

• Use appropriate molecular weight markers (30-50 kDa range)

• Include positive control samples (e.g., HepG2 whole cell lysates)

• Consider running gradient gels (5-20% SDS-PAGE) for optimal resolution

• Allow sufficient separation time (2-3 hours at 70V/90V for stacking/resolving gel)

For ELISA applications, this molecular weight information helps in assay design and ensuring that the antibody captures the full-length protein rather than degradation products.

What experimental controls should be included when using FOXA3 Antibody, HRP conjugated?

Rigorous experimental design requires appropriate controls for accurate interpretation:

• Positive tissue/cell controls:

  • HepG2 cells (high FOXA3 expression)

  • Liver tissue (mouse, rat, or human)

  • Colon and pancreatic cancer tissues

• Negative controls:

  • Isotype control (rabbit IgG at the same concentration)

  • Tissues known to have low FOXA3 expression

  • Secondary antibody-only controls (for non-HRP conjugated applications)

  • Blocking peptide competition assay to verify specificity

• Knockdown/knockout validation:

  • siRNA-mediated FOXA3 knockdown samples

  • CRISPR/Cas9-generated FOXA3 knockout cells

• Cross-reactivity assessment:

  • Test in species other than those validated (human, mouse, rat)

  • Check for cross-reactivity with other FOXA family members (FOXA1, FOXA2)

Implementing these controls helps distinguish specific signal from background and validates antibody performance in your experimental system.

How can FOXA3 Antibody be used to investigate chromatin remodeling functions?

FOXA3's role as a pioneer transcription factor makes it particularly relevant for chromatin structure research. Here are methodological approaches:

• Chromatin Immunoprecipitation (ChIP) assays:

  • For HRP-conjugated antibodies, enzymatic cleavage of HRP may be necessary before immunoprecipitation

  • Alternative: Use non-conjugated FOXA3 antibodies specifically validated for ChIP

  • Target analysis: FOXA3 binding sites on genes like AFP, albumin, G6PC1, CYP3A4, and CYP3A7

• Sequential ChIP (Re-ChIP):

  • Investigate co-occupancy of FOXA3 with other factors (e.g., CEBPA on CYP3A4 promoter)

  • Requires careful optimization of elution conditions

• ChIP-sequencing (ChIP-seq):

  • Genome-wide mapping of FOXA3 binding sites

  • Requires high-quality antibodies with minimal background

  • Data analysis should focus on motif enrichment and correlation with DNase hypersensitivity

• ATAC-seq combined with FOXA3 manipulation:

  • Compare chromatin accessibility before and after FOXA3 knockdown/overexpression

  • Can reveal genomic regions dependent on FOXA3 for accessibility

• Microscopy techniques:

  • Immunofluorescence co-localization with histone marks

  • High-resolution imaging to study FOXA3 dynamics during chromatin opening

These approaches can provide insight into how FOXA3 functions as a pioneer factor in opening chromatin structure.

What methodologies work best for studying FOXA3's role in hepatoblastoma?

Recent research has implicated FOXA3 in hepatoblastoma development . To investigate this connection:

• FOXA3 knockdown experiments:

  • siRNA approach (si-FOXA3-1, si-FOXA3-2) to reduce expression

  • Measure effects on cell viability using CCK-8 assay

  • Assess colony formation capability

• Downstream target analysis:

  • Monitor expression changes in AFP, HNF1A, MYC, and ZFHX3 following FOXA3 knockdown

  • Western blot and qRT-PCR for protein and mRNA levels, respectively

  • Immunohistochemistry in tissue samples

• Pathway analysis:

  • Investigate relationship between FOXA3 and AFP/HNF1A/MYC upregulation

  • Explore mechanisms of ZFHX3 downregulation

  • Consider chromatin accessibility at these gene loci

• Clinical correlation studies:

  • Compare FOXA3 expression levels between HB tissues and adjacent normal tissues

  • Correlate expression with clinical parameters and patient outcomes

  • Consider FOXA3 as a potential biomarker

• In vivo models:

  • Develop xenograft models with FOXA3 manipulation

  • Use FOXA3 Antibody, HRP conjugated for IHC analysis of tumor sections

These approaches can help elucidate FOXA3's mechanistic contribution to hepatoblastoma development.

How can I optimize protocols for detecting FOXA3 in different tissue types using IHC?

Immunohistochemistry optimization requires tissue-specific considerations:

• Antigen retrieval methods:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) works well for liver, colon, and pancreatic tissues

  • Compare with citrate buffer (pH 6.0) for tissues with different fixation characteristics

• Tissue-specific parameters:

  Tissue Type	Recommended Antibody Concentration	Incubation Time	Antigen Retrieval	Background Reduction
  Liver	2 μg/ml	Overnight, 4°C	EDTA, pH 8.0	10% goat serum
  Colon	2 μg/ml	Overnight, 4°C	EDTA, pH 8.0	10% goat serum
  Pancreas	2 μg/ml	Overnight, 4°C	EDTA, pH 8.0	10% goat serum

• Detection system optimization:

  • For HRP-conjugated antibodies: Direct detection with DAB substrate

  • For unconjugated antibodies: Use species-appropriate detection system (e.g., HRP Conjugated Rabbit IgG Super Vision Assay Kit)

  • Secondary antibody incubation: 30 minutes at 37°C

• Counterstaining considerations:

  • Light hematoxylin counterstain to avoid masking nuclear FOXA3 staining

  • Adjust counterstaining time based on tissue type

• Troubleshooting strategies:

  • For high background: Increase blocking time/concentration or try different blocking agents

  • For weak signal: Extend primary antibody incubation or adjust concentration

  • For non-specific binding: Additional washing steps or higher detergent concentration

These optimization steps ensure reliable FOXA3 detection across different tissue contexts.

How should I validate FOXA3 knockdown efficiency in functional studies?

When studying FOXA3 function through knockdown approaches:

• Multi-level validation approach:

  • mRNA level: qRT-PCR with FOXA3-specific primers

  • Protein level: Western blot using FOXA3 Antibody

  • Functional level: Reporter assays for FOXA3 target genes (AFP, G6PC)

• Experimental design recommendations:

  • Include multiple siRNA constructs (e.g., si-FOXA3-1, si-FOXA3-2)

  • Use appropriate negative controls (si-NC, non-targeting siRNA)

  • Establish time course of knockdown (24h, 48h, 72h)

  • Determine optimal siRNA concentration

• Quantitative assessment:

  • Calculate knockdown efficiency as percentage reduction vs. control

  • Ensure at least 70-80% knockdown for functional studies

  • Correlate knockdown level with phenotypic changes

• Addressing potential confounding factors:

  • Check for compensatory upregulation of related factors (FOXA1, FOXA2)

  • Verify specificity using rescue experiments (re-expression of siRNA-resistant FOXA3)

  • Consider off-target effects through transcriptome analysis

• Integration with functional assays:

  • Cell viability (CCK-8 assay)

  • Colony formation ability

  • Expression of known FOXA3 targets (AFP, HNF1A, ZFHX3, MYC)

This comprehensive validation ensures that observed phenotypes are truly attributable to FOXA3 knockdown.

How can I study FOXA3's interactions with co-factors in transcriptional regulation?

FOXA3 cooperates with various co-factors to regulate gene expression:

• Co-immunoprecipitation (Co-IP) techniques:

  • Use FOXA3 Antibody to pull down FOXA3 and associated proteins

  • For HRP-conjugated antibodies: Consider enzymatic cleavage of HRP before IP

  • Western blot for known interactors (CEBPA for CYP3A4 regulation, CTF/NF-I family for CYP3A7)

  • Mass spectrometry for unbiased identification of novel interactors

• Proximity ligation assay (PLA):

  • In situ detection of FOXA3 interactions with suspected partners

  • Provides spatial information about interaction in cellular context

  • Requires specific antibodies for both FOXA3 and its partner(s)

• Reporter gene assays:

  • Construct reporters containing FOXA3 binding sites (e.g., G6PC, CYP3A4 promoters)

  • Co-transfect with FOXA3 and potential co-factors

  • Measure additive or synergistic effects on transcriptional activity

• DNA-protein complex analysis:

  • Electrophoretic mobility shift assay (EMSA) with purified factors

  • Supershift assays using FOXA3 Antibody

  • DNA pulldown followed by Western blot for co-binding factors

• Chromatin studies:

  • Sequential ChIP to identify co-occupancy at genomic loci

  • Manipulation of co-factor levels to assess impact on FOXA3 binding

These approaches provide a comprehensive view of FOXA3's cooperative interactions in transcriptional regulation.

What are common challenges when working with FOXA3 Antibody, HRP conjugated and how can they be addressed?

Researchers may encounter several technical issues:

• High background in immunoassays:

  • Cause: Insufficient blocking or non-specific binding

  • Solution: Increase blocking agent concentration (10% goat serum recommended)

  • Alternative: Try different blocking agents (BSA, casein, commercial blockers)

  • Additional: Include 0.1-0.3% Tween-20 in wash buffers

• Weak or absent signal:

  • Cause: Insufficient antigen, degraded antibody, or suboptimal conditions

  • Solution: Verify protein expression in positive control (HepG2 cells)

  • Optimize: Increase antibody concentration or incubation time

  • Consider: Different antigen retrieval methods for IHC/ICC

• Multiple bands in Western blot:

  • Cause: Degradation products, isoforms, or cross-reactivity

  • Verification: Compare with literature-reported patterns

  • Solution: Use freshly prepared samples with protease inhibitors

  • Validation: Confirm specificity with knockdown samples

• Reduced HRP activity:

  • Cause: Repeated freeze-thaw cycles or improper storage

  • Prevention: Prepare small working aliquots

  • Testing: Verify HRP activity with substrate-only control

  • Alternative: Consider using fresh antibody for critical experiments

• Signal variability between experiments:

  • Cause: Inconsistent technique or reagent degradation

  • Solution: Standardize protocols and reagent preparation

  • Control: Include internal standards for normalization

  • Recommendation: Prepare master mixes where possible

Addressing these challenges systematically improves reproducibility and data quality.

How should I interpret discrepancies between FOXA3 mRNA and protein expression data?

Differences between mRNA and protein levels are common for transcription factors and require careful interpretation:

• Biological explanations:

  • Post-transcriptional regulation (miRNAs, RNA-binding proteins)

  • Post-translational modifications affecting protein stability

  • Feedback mechanisms regulating FOXA3 levels

  • Different half-lives of mRNA versus protein

• Technical considerations:

  • Antibody specificity and sensitivity

  • RNA quality and reverse transcription efficiency

  • Different dynamic ranges of detection methods

  • Subcellular localization affecting protein detection

• Validation approaches:

  • Time-course analysis to detect temporal relationships

  • Inhibitor studies (proteasome, translation inhibitors)

  • Alternative detection methods (mass spectrometry)

  • Single-cell analysis to assess population heterogeneity

• Data integration framework:

  Observation	Possible Explanation	Validation Approach
  High mRNA, low protein	Rapid protein degradation	Proteasome inhibition
  Low mRNA, high protein	Protein stability, sensitivity differences	Pulse-chase labeling
  Changing ratios across conditions	Condition-specific regulation	Time course analysis
  Tissue-specific discrepancies	Tissue-specific post-transcriptional control	Multi-tissue comparison

• Research context:

  • In hepatoblastoma research, consider disease-specific alterations in FOXA3 regulation

  • For liver gene expression studies, examine relationships with FOXA3 targets (AFP, albumin)

Acknowledging these complexities enables more accurate interpretation of experimental data involving FOXA3 expression.

How can FOXA3 Antibody be used to investigate metabolic disease mechanisms?

FOXA3's role in glucose homeostasis makes it relevant for metabolic disease research:

• Tissue-specific expression analysis:

  • Compare FOXA3 levels in normal versus diabetic liver tissue

  • Correlation with glucose-regulating genes (G6PC1, PEPCK)

  • Relationship with insulin signaling pathway components

• Glucose regulation studies:

  • FOXA3 binding to G6PC promoter under different glycemic conditions

  • Effects of insulin/glucagon on FOXA3 expression and activity

  • FOXA3 knockdown impact on glucose production in hepatocytes

• Nutritional intervention models:

  • FOXA3 expression changes during fasting/feeding cycles

  • Response to high-fat diet or ketogenic diet

  • Integration with other metabolic transcription factors

• Therapeutic target assessment:

  • Small molecule screening for FOXA3 activity modulation

  • Analysis of FOXA3 binding site polymorphisms in metabolic disease cohorts

  • Correlation of FOXA3 expression with response to anti-diabetic treatments

• Multi-omics integration:

  • Correlation of FOXA3 chromatin binding with metabolomic profiles

  • Proteomic analysis of FOXA3 interactome in metabolic disease states

  • Integration with genome-wide association studies (GWAS) data

These approaches can reveal FOXA3's contributions to metabolic disease pathogenesis and potential therapeutic relevance.

What are the latest methodologies for studying FOXA3's role in cancer development beyond hepatoblastoma?

While FOXA3's role in hepatoblastoma is established , its function in other cancers is emerging:

• Cancer-type specific expression profiling:

  • Use FOXA3 Antibody, HRP conjugated for tissue microarray analysis

  • Compare expression across cancer types and stages

  • Correlate with patient outcomes and treatment response

• Mechanistic studies:

  • FOXA3's interaction with tumor suppressor genes (e.g., ZFHX3)

  • Relationship with oncogenes (e.g., MYC)

  • Effects on cancer stemness and differentiation markers

• Epigenetic landscape analysis:

  • FOXA3 binding at cancer-relevant gene promoters

  • Correlation with DNA methylation patterns

  • Histone modification changes at FOXA3 binding sites

• Drug response modulation:

  • FOXA3 expression changes following chemotherapy/targeted therapy

  • Impact of FOXA3 knockdown on drug sensitivity

  • Combination approaches targeting FOXA3 networks

• Emerging technologies:

  • Single-cell analysis of FOXA3 expression heterogeneity within tumors

  • Spatial transcriptomics to map FOXA3 expression in tumor microenvironment

  • CRISPR screens to identify synthetic lethal interactions with FOXA3

These methodologies expand our understanding of FOXA3's roles beyond liver cancer to other malignancies where pioneer transcription factors contribute to disease progression.