FOXA3 Antibody, Biotin conjugated

What is FOXA3 and why is it important in research?

FOXA3 (Forkhead box protein A3) is a transcription factor also known as Hepatocyte nuclear factor 3-gamma (HNF-3-gamma), HNF-3G, Fork head-related protein FKH H3, or Transcription factor 3G (TCF-3G) . It functions as a key regulator in gene expression, particularly in liver development and metabolism. FOXA3 has significant importance in research due to its role as a pioneer transcription factor that can access condensed chromatin and facilitate the binding of other transcription factors. FOXA3 is particularly important in studies related to liver development, metabolism regulation, and certain cancer types, including hepatoblastoma. Recent research has demonstrated that FOXA3 regulates ApoA-I expression and macrophage reverse cholesterol transport (RCT), highlighting its importance in lipid metabolism studies .

What is the specificity of the FOXA3 Antibody, Biotin conjugated?

The FOXA3 Antibody, Biotin conjugated is designed to recognize human FOXA3 protein. It is a polyclonal antibody raised in rabbit using recombinant Human Hepatocyte nuclear factor 3-gamma protein (specifically amino acids 220-336) as the immunogen . The antibody has been tested for reactivity with human samples. As with all antibodies, cross-reactivity with highly homologous proteins (such as other FOXA family members) should be considered and validated experimentally when working in critical research applications. Researchers should perform appropriate controls, particularly when studying systems where multiple FOXA family members are expressed simultaneously.

How should FOXA3 Antibody, Biotin conjugated be incorporated into EMSA experiments?

For Electrophoretic Mobility Shift Assay (EMSA) experiments using FOXA3 Antibody, Biotin conjugated, researchers should follow a protocol similar to that described in the literature. First, synthesize and label 50 base pair oligonucleotides containing putative FOXA3 binding sites with biotin on the 3′ end following manufacturer's instructions . Prepare cell lysates overexpressing FOXA3 (such as from 293A cells) for incubation with the probes.

For competition studies, include unlabeled oligonucleotides as controls: wild-type oligos (containing putative FOXA3 binding sites), and mutated versions (with site 1 mutation, site 2 mutation, or both sites mutated) . For supershift assays, incubate cell lysates with the probe first, followed by incubation with the FOXA3 antibody, using the same amount of IgG as a negative control. The biotin conjugation of the antibody should be considered when designing detection methods, as it may influence binding characteristics or detection protocols compared to unconjugated antibodies .

What considerations should be made when using FOXA3 Antibody, Biotin conjugated for immunohistochemistry?

When using FOXA3 Antibody, Biotin conjugated for immunohistochemistry, several technical considerations should be addressed. Based on published protocols, tissue sections should be dewaxed and treated with 3% hydrogen peroxide to inactivate endogenous peroxidases . Antigen retrieval using sodium citrate buffer (pH 6.0) and microwave heating is recommended. For blocking, 10% normal goat serum is typically used .

The antibody dilution requires optimization; literature suggests starting with a 1:150 dilution for FOXA3 antibodies . Since the antibody is already biotin-conjugated, the detection system should be streptavidin-based rather than using biotin-labeled secondary antibodies. Be aware of potential endogenous biotin in tissues (particularly liver), which may require additional blocking steps. FOXA3 is predominantly localized in the nucleus, so nuclear staining patterns should be expected and evaluated . Include appropriate positive and negative controls, and consider dual staining with other markers (like AFP) for co-localization studies in research contexts such as hepatoblastoma.

How can I validate the specificity of results when using FOXA3 Antibody, Biotin conjugated?

To validate the specificity of results when using FOXA3 Antibody, Biotin conjugated, implement multiple control measures. First, include a negative control by using non-immune rabbit IgG at the same concentration as the primary antibody to assess non-specific binding . For positive controls, use tissues or cell lines known to express FOXA3, such as liver tissues or hepatoblastoma cells based on published literature .

Validation can also be strengthened through competitive blocking experiments, where pre-incubation of the antibody with its specific immunogen peptide should abolish specific staining. Additionally, perform parallel experiments using alternative antibodies against FOXA3 from different sources or against different epitopes. For functional validation, correlate antibody staining with other techniques such as RT-PCR, RNA-seq, or knockdown/overexpression studies that measure FOXA3 at the transcript level . Finally, the pattern of staining should be consistent with the expected subcellular localization of FOXA3 (predominantly nuclear) and should correlate with known biological functions and expression patterns in your experimental system.

How can FOXA3 Antibody, Biotin conjugated be used to study the role of FOXA3 in hepatoblastoma progression?

To investigate FOXA3's role in hepatoblastoma (HB) progression using the FOXA3 Antibody, Biotin conjugated, researchers can implement several sophisticated approaches. First, perform immunohistochemistry or immunofluorescence on paired HB tissues and adjacent normal tissues to quantify FOXA3 expression levels and localization patterns . The biotin-conjugated format is particularly useful for amplifying detection signals in tissue samples with potentially low expression levels.

For mechanistic studies, combine the antibody with chromatin immunoprecipitation (ChIP) assays to identify FOXA3 binding sites within regulatory regions of target genes implicated in HB (like AFP, HNF1A, and ZFHX3) . This can be followed by reporter gene assays to validate the functionality of these interactions. In cellular models, use the antibody to monitor changes in FOXA3 expression and localization following genetic manipulations (knockdown or overexpression) and correlate these with phenotypic changes in proliferation, colony formation, and expression of downstream targets .

Co-immunoprecipitation experiments using the biotin-conjugated antibody can help identify protein-protein interaction partners of FOXA3 in HB cells, potentially revealing novel regulatory mechanisms. Finally, the antibody can be used in tissue microarray analysis of large cohorts of HB samples to correlate FOXA3 expression with clinical parameters, patient outcomes, and molecular subtypes, providing translational insights into the prognostic value of FOXA3 in HB .

What are the considerations for using FOXA3 Antibody, Biotin conjugated in studying FOXA3's interaction with chromatin?

Studying FOXA3's interaction with chromatin using FOXA3 Antibody, Biotin conjugated requires careful experimental design. While direct information about FOXA3's chromatin interactions is limited in the provided search results, insights can be derived from studies of the related family member FOXA1 . For ChIP experiments, standard chromatin preparation protocols may need modification as pioneer factors like FOXA proteins can bind condensed chromatin. Formaldehyde crosslinking times may need optimization to capture these interactions effectively.

For more comprehensive understanding, combine ChIP-seq with ATAC-seq or DNase-seq to correlate FOXA3 binding with changes in chromatin accessibility. Additionally, consider the potential influence of post-translational modifications on FOXA3's interaction with chromatin, as studies with FOXA1 have revealed that small molecule interactions with specific residues can alter DNA binding properties and genomic localization patterns .

How can FOXA3 Antibody, Biotin conjugated be utilized to investigate the regulatory relationship between FOXA3 and AFP in liver diseases?

To investigate the regulatory relationship between FOXA3 and Alpha-fetoprotein (AFP) in liver diseases using FOXA3 Antibody, Biotin conjugated, researchers should implement a multi-faceted approach. Begin with dual immunostaining or sequential immunohistochemistry on liver disease tissues (such as hepatoblastoma or hepatocellular carcinoma) to assess co-expression patterns of FOXA3 and AFP . The biotin-conjugated antibody allows for flexible detection strategies when combined with differently labeled AFP antibodies.

For mechanistic studies, use the antibody in ChIP assays followed by qPCR targeting the AFP promoter region to quantify FOXA3 occupancy. This can be complemented with reporter gene assays using luciferase constructs containing the AFP promoter, with or without mutations in putative FOXA3 binding sites. In cellular models, combine the antibody with knockdown or overexpression studies of FOXA3 to monitor consequent changes in AFP expression at both protein (using Western blot) and mRNA levels (using RT-qPCR) .

For more comprehensive analysis, perform ChIP-seq to identify genome-wide binding patterns of FOXA3 in relation to AFP and other target genes. This should be integrated with RNA-seq data following FOXA3 manipulation to establish direct versus indirect regulatory relationships. Finally, examine how this regulatory relationship changes under different disease states or experimental conditions, such as hypoxia or inflammatory stimuli, which are known to influence both FOXA3 and AFP expression in liver pathologies .

What is the optimal storage and handling protocol for FOXA3 Antibody, Biotin conjugated?

The optimal storage and handling protocol for FOXA3 Antibody, Biotin conjugated requires careful attention to manufacturer recommendations. Upon receipt, the antibody should be stored at -20°C or -80°C . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of antibody activity. The antibody is supplied in liquid form with a buffer composition of 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

For handling, aliquot the antibody into smaller volumes based on anticipated usage to minimize freeze-thaw cycles. When thawing, allow the antibody to thaw completely at 4°C before use and avoid room temperature exposure for extended periods. Centrifuge briefly before opening to ensure all liquid is at the bottom of the tube. For dilution, use buffers compatible with the storage buffer, typically PBS with BSA or non-fat dry milk as carriers. The biotin conjugation may be sensitive to certain buffer components, so avoid buffers containing sodium azide or other preservatives that could interfere with the biotin-streptavidin interaction. Document all freeze-thaw cycles and perform periodic validation tests to ensure antibody performance has not deteriorated over time.

What is the recommended protocol for using FOXA3 Antibody, Biotin conjugated in immunofluorescence studies?

For immunofluorescence studies using FOXA3 Antibody, Biotin conjugated, adapt the following protocol based on published methods. Begin with standard tissue section preparation, including appropriate fixation (typically 4% paraformaldehyde) and permeabilization steps. Wash the sections thoroughly with PBS buffer (five times, 10 minutes each) . Block with 10% normal goat serum at 37°C for 90 minutes to reduce non-specific binding.

Incubate the sections with appropriately diluted FOXA3 Antibody, Biotin conjugated at 4°C for 48 hours . After incubation, wash again with PBS buffer (five times, 10 minutes each). Since the antibody is already biotin-conjugated, follow with streptavidin-conjugated fluorophore incubation (e.g., streptavidin-Alexa Fluor 488 or 594) according to manufacturer's recommended concentration and time. Counterstain nuclei with DAPI staining solution at room temperature for 5 minutes .

Wash with PBS three more times, then mount the slides using anti-fluorescence quenching mounting medium. For co-localization studies with other proteins (such as AFP), consider sequential immunostaining or simultaneous staining with antibodies from different host species. Image using an inverted fluorescence microscope with appropriate filter sets for the chosen fluorophores . Include controls for autofluorescence, non-specific binding, and cross-reactivity. FOXA3 should primarily show nuclear localization, which can serve as an internal validation of staining specificity.

How should dilution and titration of FOXA3 Antibody, Biotin conjugated be determined for different applications?

Determining optimal dilution and titration for FOXA3 Antibody, Biotin conjugated requires systematic testing across different applications. For ELISA, the validated application for this antibody , start with a dilution range of 1:500 to 1:5000 and perform a standard curve analysis to determine the optimal working dilution that provides the highest signal-to-noise ratio while remaining in the linear range of detection.

For immunohistochemistry applications, though not explicitly validated for this particular conjugate, literature suggests starting with a dilution of approximately 1:150 for FOXA3 antibodies . Prepare a dilution series (e.g., 1:50, 1:100, 1:150, 1:200, 1:500) and test on positive control tissues known to express FOXA3. Evaluate staining intensity, specificity, and background levels to determine the optimal dilution.

For Western blot applications, if attempted, begin with dilutions of 1:500 to 1:2000 and include both positive and negative control samples. For immunofluorescence, start with dilutions similar to immunohistochemistry but be aware that the biotin-streptavidin detection system may require further optimization of secondary reagent concentrations. In all applications, consider the concentration of the antibody (typically provided in μg/μL) rather than just the dilution factor for more reproducible results between different antibody lots. Document optimal conditions thoroughly, including incubation times and temperatures, which can significantly impact antibody performance across different applications.

How does FOXA3 expression correlate with clinical outcomes in hepatoblastoma, and how can the antibody be used to investigate this?

FOXA3 expression has been found to be significantly upregulated in hepatoblastoma (HB) tissues compared to normal liver tissues, suggesting its potential role as a biomarker for this pediatric liver cancer . Research indicates that FOXA3 may promote the occurrence and development of HB by upregulating AFP and HNF1A/MYC expression while downregulating ZFHX3 expression . To investigate correlations between FOXA3 expression and clinical outcomes, researchers can use FOXA3 Antibody, Biotin conjugated in immunohistochemistry studies on tissue microarrays from large cohorts of HB patients with documented clinical follow-up.

The methodology should include standardized scoring systems for nuclear FOXA3 staining intensity and percentage of positive cells. These scores can then be correlated with clinicopathological parameters including tumor stage, histological subtype, metastatic status, and patient survival data using appropriate statistical analyses. The biotin-conjugated format offers advantages for signal amplification in archival tissue samples. Additionally, researchers can perform double staining with markers of proliferation (Ki-67) or other prognostic indicators to develop more sophisticated predictive models.

For functional validation, the antibody can be used to confirm FOXA3 expression levels in patient-derived xenograft models or primary cell cultures from HB patients with different clinical outcomes. This approach allows for connecting ex vivo molecular findings with in vivo behavior and patient outcomes, potentially identifying FOXA3 as a therapeutic target in aggressive forms of HB .

What insights can be gained about FOXA3's role in gene regulation by combining the antibody with genomic techniques?

Combining FOXA3 Antibody, Biotin conjugated with genomic techniques offers powerful insights into FOXA3's role in gene regulation. The biotin conjugation is particularly advantageous for ChIP-seq applications, where the antibody can be used to precipitate FOXA3-bound chromatin fragments, followed by next-generation sequencing to identify genome-wide binding sites . This approach can reveal FOXA3's binding motif preferences, distal versus proximal binding patterns, and co-localization with other transcription factors.

Integrating ChIP-seq data with RNA-seq from the same biological samples can establish direct correlations between FOXA3 binding and gene expression changes. For instance, in hepatoblastoma research, this combination has helped identify that FOXA3 regulates genes including AFP, HNF1A, and influences the MYC pathway . The antibody can also be used in CUT&RUN or CUT&Tag assays, which offer higher resolution and lower background than traditional ChIP-seq.

For studying FOXA3's pioneer factor activity, combining the antibody with ATAC-seq before and after FOXA3 manipulation can demonstrate its role in chromatin accessibility changes. Additionally, HiChIP or ChIA-PET approaches using the biotin-conjugated antibody can reveal long-range chromatin interactions mediated by FOXA3, providing insights into its role in three-dimensional genome organization. These multi-omics approaches have revealed that transcription factors like FOXA3 often function within complex regulatory networks, with context-dependent binding patterns that vary across cell types and disease states .

How can contradictory findings about FOXA3 function be reconciled through careful antibody-based experiments?

Contradictory findings about FOXA3 function can be reconciled through carefully designed antibody-based experiments that address potential sources of discrepancy. First, use the FOXA3 Antibody, Biotin conjugated to verify FOXA3 expression levels and localization patterns across the specific cellular contexts being compared, as FOXA3 function may be highly context-dependent. When contradictory results appear in literature, confirm that the detected protein is indeed FOXA3 by complementing antibody-based detection with orthogonal methods such as mass spectrometry or mRNA analysis.

For conflicting reports on FOXA3's regulatory targets, perform ChIP-seq experiments under standardized conditions across different cell types or disease states to determine if binding patterns change contextually. Compare these binding patterns with chromatin accessibility data and expression of target genes to establish direct versus indirect regulatory relationships. The biotin conjugation allows for efficient pull-down in these applications.

To address contradictions regarding FOXA3's role in disease progression, conduct time-course experiments using the antibody to track FOXA3 expression and localization changes throughout disease development. In hepatoblastoma studies, for example, seemingly opposing roles of FOXA3 might be explained by its differential effects on distinct downstream pathways (e.g., HNF1A/MYC activation versus ZFHX3 suppression) . Finally, use the antibody in co-immunoprecipitation experiments followed by mass spectrometry to identify FOXA3's protein interaction partners across different experimental conditions, as these interactions may modulate its function and contribute to apparently contradictory observations in different research contexts.