HNF4A Recombinant Monoclonal Antibody

What is HNF4A and why is it an important research target?

Hepatocyte nuclear factor 4 alpha (HNF4A) is an orphan nuclear receptor that functions as a transcription factor. It plays crucial roles in the development and function of several tissues, particularly the liver, kidney, intestine, and pancreas. HNF4A binds to DNA exclusively as a homodimer and regulates numerous genes involved in metabolism and development . The importance of HNF4A in research stems from its involvement in several pathological conditions, most notably maturity-onset diabetes of the young type 1 (MODY1), which is associated with mutations in the HNF4A gene . Additionally, recent research has identified roles for HNF4A in sepsis-associated lung injury and potential associations with type 2 diabetes in certain populations . The HNF4A gene undergoes alternative splicing, generating up to nine different isoforms (HNF4a1 through HNF4a9), which adds complexity to its study and makes specific antibody detection particularly valuable .

What are the key advantages of using recombinant rabbit monoclonal antibodies for HNF4A detection?

Recombinant rabbit monoclonal antibodies offer several significant advantages over traditional antibodies for HNF4A detection in research applications:

These advantages arise from the production method, which involves cloning specific antibody DNA sequences from immunoreactive rabbits and screening individual clones to select optimal candidates for production . This approach yields antibodies with consistent performance characteristics essential for reliable research outcomes.

What applications are HNF4A recombinant monoclonal antibodies suitable for?

HNF4A recombinant monoclonal antibodies have been validated for multiple research applications, making them versatile tools for studying this transcription factor. Based on validation studies, these antibodies can be effectively used in:

• Flow Cytometry: For quantitative analysis of HNF4A expression in single cells

• Immunocytochemistry (ICC): For visualization of HNF4A in cultured cells

• Immunohistochemistry (IHC): For detection of HNF4A in tissue sections

• Western Blot: For protein expression analysis and molecular weight determination

Researchers should verify the specific applications validated for each antibody clone, as performance can vary. For example, the SN72-03 recombinant rabbit monoclonal antibody has been validated for all these applications, while other clones may have different validated use cases .

What is the recommended protocol for using HNF4A antibodies in immunohistochemistry on paraffin-embedded tissues?

For optimal results when using HNF4A recombinant monoclonal antibodies on paraffin-embedded tissue sections, researchers should follow this methodological approach:

• Fixation: Use paraformaldehyde (PFA) for tissue fixation, as it provides superior tissue penetration compared to alternative fixatives. Importantly, PFA should be freshly prepared before use, as long-term stored PFA converts to formalin as the molecules aggregate .

• Antigen Retrieval: Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). The optimal retrieval buffer may vary based on the specific antibody clone and should be determined empirically.

• Blocking: Block with 5-10% normal serum (from the same species as the secondary antibody) in PBS containing 0.1-0.3% Triton X-100 for 1-2 hours at room temperature.

• Primary Antibody Incubation: Dilute the HNF4A antibody in antibody diluent (typically 1:100 to 1:500, but optimal dilution should be determined experimentally) and incubate overnight at 4°C.

• Detection System: Use an appropriate detection system compatible with rabbit primary antibodies, such as polymer-based detection systems or biotinylated secondary antibodies followed by streptavidin-HRP.

• Controls: Always include both positive controls (tissues known to express HNF4A, such as liver) and negative controls (omitting primary antibody) to validate staining specificity .

This protocol has been demonstrated to produce consistent and specific staining of HNF4A in liver and kidney tissues, which are known to express this transcription factor .

How can researchers confirm the specificity of HNF4A antibody binding in their experimental system?

Confirming antibody specificity is crucial for reliable research results. For HNF4A recombinant monoclonal antibodies, researchers should implement multiple validation approaches:

• Positive and Negative Tissue Controls: Test the antibody on tissues known to express HNF4A (positive controls like liver and kidney) and tissues expected to lack expression (negative controls). Compare staining patterns with published literature reports .

• Multiple Detection Methods: Validate binding using complementary techniques such as immunofluorescence, western blot, and flow cytometry, as demonstrated in recent studies that confirmed HNF4A antibody specificity across these methods .

• Peptide Competition Assay: Pre-incubate the antibody with the immunogen peptide before application to samples. Specific binding should be blocked by the peptide, resulting in diminished or absent signal.

• Genetic Controls: Where possible, use cells or tissues with HNF4A knockdown/knockout or overexpression to confirm signal correlation with expression levels.

• Cross-Validation with Different Antibody Clones: Compare results using different HNF4A antibodies targeting distinct epitopes. For example, researchers have expanded their HNF4A target lists by using different antibodies (such as ab41898) in ChIP-Seq experiments .

• Correlation with mRNA Expression: Compare protein detection patterns with HNF4A mRNA expression data from RT-PCR or RNA-seq to ensure concordance.

Implementation of these complementary validation strategies provides robust evidence for antibody specificity and strengthens the reliability of research findings involving HNF4A detection .

What are the optimal storage and handling conditions for maintaining HNF4A antibody performance over time?

Proper storage and handling of HNF4A recombinant monoclonal antibodies is essential for maintaining their performance characteristics. The following guidelines should be followed:

• Long-term Storage: Store unopened antibody at -20°C for up to one year. Most HNF4A antibody formulations contain stabilizers like glycerol (typically 40%) that prevent freeze damage .

• Short-term Storage: For frequent use within a one-month period, store at 4°C to avoid repeated freeze-thaw cycles .

• Avoid Freeze-Thaw Cycles: Minimize freeze-thaw events, as they can lead to protein denaturation, aggregation, and loss of activity. If possible, prepare small aliquots before freezing .

• Buffer Composition: Most commercially available HNF4A antibodies are supplied in TBS with stabilizers such as BSA (0.05%), glycerol (40%), and preservatives like sodium azide (0.05%) at pH 7.4. This formulation helps maintain antibody integrity during storage .

• Temperature Transitions: When thawing frozen antibody, allow it to thaw completely at 4°C rather than at room temperature or with artificial heating.

• Centrifugation: Briefly centrifuge the antibody vial before opening to collect all liquid at the bottom of the vial and reduce loss.

• Contamination Prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination.

Following these recommendations can significantly extend the useful life of HNF4A antibodies and ensure consistent experimental results over time.

How can HNF4A antibodies be optimized for chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation with HNF4A antibodies requires careful optimization to achieve high specificity and sensitivity. The following methodological approach is recommended based on successful ChIP-Seq studies:

• Antibody Selection: Choose antibodies validated specifically for ChIP applications. In published studies, researchers have used multiple HNF4A antibodies (including ab41898) to expand their target list coverage in ChIP-Seq experiments .

• Cross-Validation: Compare results from at least two different HNF4A antibody clones targeting distinct epitopes to confirm binding sites. This approach has been used to expand HNF4A target lists in HepG2 cells, capturing approximately half of the unique HNF4A-bound gene targets reported in ENCODE .

• Fixation Optimization: Titrate formaldehyde concentration (typically 0.75-1%) and fixation time (typically 10-15 minutes) based on the specific cell type being studied.

• Sonication Parameters: Optimize sonication conditions to generate chromatin fragments of 200-500 bp, which is optimal for HNF4A ChIP-Seq analyses.

• Antibody Concentration: Typically, 2-5 μg of HNF4A antibody per ChIP reaction is used, but this should be titrated for each experimental system.

• Controls:

  • Input control: Reserved chromatin before immunoprecipitation

  • IgG control: Non-specific IgG from the same species as the HNF4A antibody

  • Positive control regions: Known HNF4A binding sites such as the HNF1A promoter, as HNF4A is known to bind the HNF1A promoter region

• Sequential ChIP: For studies of co-binding with other transcription factors, sequential ChIP can be performed using the HNF4A antibody followed by antibodies against potential binding partners.

By implementing these optimizations, researchers can achieve high-quality ChIP-Seq data for HNF4A binding sites, as demonstrated in studies that have successfully identified genome-wide HNF4A binding patterns in various cell types .

What strategies can be employed to study tissue-specific HNF4A isoform expression?

HNF4A exists in multiple isoforms (HNF4a1 through HNF4a9) with tissue-specific expression patterns. Studying these isoforms requires specialized approaches:

• Isoform-Specific Antibodies: Carefully select antibodies that can distinguish between HNF4A isoforms based on their epitope location. Not all HNF4A antibodies can differentiate between isoforms, so epitope mapping is essential .

• Western Blot Analysis: Use high-resolution SDS-PAGE (10-12%) to separate closely related isoforms based on slight molecular weight differences. Extended running times and gradient gels may improve separation.

• RT-PCR with Isoform-Specific Primers: Design primers that span unique exon junctions or target isoform-specific sequences to quantify isoform-specific mRNA expression across tissues.

• Temporal Analysis During Development: Analyze expression at different developmental stages, as isoform expression changes during tissue development. For example, in pancreatic differentiation, studies have examined HNF4A expression in day 14 pancreatic progenitors, day 20 endocrine progenitors, and day 35 beta-like cells to capture developmental changes .

• Cell Type-Specific Analysis: Use techniques like laser capture microdissection or single-cell RNA-seq to isolate specific cell populations within heterogeneous tissues for isoform analysis.

• Dual Immunofluorescence: Combine HNF4A antibodies with markers for specific cell types to determine cell-specific isoform expression patterns within complex tissues.

• Recombinant Expression Controls: Generate recombinant proteins of each HNF4A isoform to serve as positive controls for antibody specificity testing.

Implementing these approaches allows researchers to precisely map the expression patterns of specific HNF4A isoforms across tissues and developmental stages, providing insights into their specialized functions .

How can researchers effectively study HNF4A in relation to diabetes pathophysiology?

HNF4A mutations are associated with maturity-onset diabetes of the young type 1 (MODY1), and variants have been implicated in type 2 diabetes susceptibility. To effectively study HNF4A in diabetes contexts:

• Genetic Association Studies: Design studies with adequate power to detect small genetic effects. In Scandinavian populations, a pooled odds ratio of 1.14 (95% CI 1.06-1.23, P=0.0004) was found for the HNF4A SNP rs1884613, highlighting the importance of large, homogeneous study populations for detecting modest genetic effects .

• Population Stratification: Consider population heterogeneity, as HNF4A associations with type 2 diabetes show significant population differences. Meta-analyses including 12,292 type 2 diabetic cases and 15,519 controls revealed significant heterogeneity between populations .

• Promoter-Specific Analysis: Target both P1 and P2 promoters of HNF4A, located 45.5 kb apart on chromosome 20q, as they regulate tissue-specific expression. Carefully design experiments to distinguish between these promoter regions .

• Functional Studies of Variants: Use reporter gene assays, EMSA, and CRISPR-based approaches to assess the functional impact of diabetes-associated HNF4A variants on transcriptional activity.

• Pancreatic β-Cell Models: Implement human pluripotent stem cell (hPSC)-derived pancreatic cell models at different developmental stages to study HNF4A function in β-cell development and function. Key stages include day 14 pancreatic progenitors, day 20 endocrine progenitors, and day 35 beta-like cells .

• Target Gene Analysis: Identify and study HNF4A target genes in pancreatic β-cells using ChIP-Seq and RNA-Seq approaches. Compare these with targets in other tissues to identify β-cell-specific regulatory networks.

• Animal Models: Utilize tissue-specific conditional knockout models to assess the impact of HNF4A deficiency on glucose homeostasis and β-cell function.

These integrated approaches enable comprehensive investigation of HNF4A's role in diabetes pathophysiology, potentially revealing novel therapeutic targets .

What are common sources of false positives/negatives when using HNF4A antibodies and how can they be mitigated?

Sources of False Positives:

• Cross-reactivity: Some antibodies may recognize proteins with similar epitopes to HNF4A. Mitigation: Use recombinant rabbit monoclonal antibodies, which offer improved specificity . Validate with multiple detection methods and knockout controls.

• Non-specific binding: High antibody concentrations can lead to non-specific binding. Mitigation: Titrate antibody concentrations and include appropriate blocking steps using 5-10% normal serum from the same species as the secondary antibody.

• Endogenous peroxidase activity: In IHC applications, endogenous peroxidases can cause background staining. Mitigation: Include hydrogen peroxide blocking steps before primary antibody application.

Sources of False Negatives:

• Epitope masking: Fixation can mask epitopes, particularly in formalin-fixed tissues. Mitigation: Optimize antigen retrieval methods; use freshly prepared PFA for fixation rather than long-stored PFA which converts to formalin .

• Antibody storage degradation: Improper storage leads to reduced antibody activity. Mitigation: Follow recommended storage conditions (-20°C for long-term, 4°C for short-term use) and avoid repeated freeze-thaw cycles .

• Isoform specificity: Some antibodies may not detect all HNF4A isoforms. Mitigation: Verify which isoforms your antibody detects by referring to the immunogen sequence information . Use multiple antibodies targeting different epitopes for comprehensive detection.

• Tissue-specific expression levels: HNF4A expression varies across tissues and developmental stages. Mitigation: Include positive control tissues known to express HNF4A (liver, kidney) in your experiments . Optimize exposure/development times for tissues with lower expression.

By implementing these mitigation strategies, researchers can significantly improve the reliability of their HNF4A detection results across various experimental applications.

How should researchers interpret HNF4A antibody signals in different cellular compartments?

HNF4A is primarily a nuclear transcription factor, but its distribution can vary depending on cellular context, experimental conditions, and detection method. Proper interpretation requires understanding these nuances:

• Nuclear Localization: In most tissues, including liver, kidney, intestine, and pancreas, HNF4A predominantly localizes to the nucleus, consistent with its function as a transcription factor. Strong nuclear staining is typically expected and represents active HNF4A .

• Cytoplasmic Signals: While typically considered an artifact, cytoplasmic HNF4A staining may represent:

  • Newly synthesized protein in transit to the nucleus

  • Post-translational modifications affecting nuclear localization

  • Non-specific antibody binding (a technical artifact)

  • Specific isoforms with altered localization

  Validation approach: Compare cytoplasmic signals across multiple antibody clones and detection methods. True cytoplasmic localization should be consistent across methods and show correlation with cellular functions.

• Nuclear-Cytoplasmic Shuttling: Under certain conditions, such as cellular stress or signaling events, HNF4A might exhibit altered localization. These changes can be physiologically relevant and should be interpreted in context.

• Punctate Nuclear Patterns: HNF4A often shows punctate patterns within the nucleus, potentially representing transcriptional complexes or binding to specific chromatin regions. This pattern is consistent with its role as a transcription factor binding to specific DNA regions.

• Context-Dependent Expression: Expression levels and patterns vary significantly across development and cell differentiation. For example, in hepatic differentiation, HNF4A is most highly expressed in hepatoblasts (D8), while in pancreatic development, expression patterns change across different stages (D14 pancreatic progenitors, D20 endocrine progenitors, D35 beta-like cells) .

Proper interpretation requires comparing signals to appropriate controls, using multiple detection methods, and considering the biological context of the experimental system being studied.

How can researchers reconcile contradictory results from different HNF4A antibody clones?

When different HNF4A antibody clones yield contradictory results, systematic troubleshooting and reconciliation strategies are needed:

• Epitope Mapping: Identify the exact epitope targeted by each antibody clone. Epitopes in different regions of HNF4A may be differentially accessible in certain contexts or may be specific to certain isoforms. For example, antibodies targeting sequences within Human HNF-4-alpha aa 21-70 will detect different isoform subsets than those targeting C-terminal regions .

• Isoform Specificity Analysis: Determine which HNF4A isoforms (HNF4a1 through HNF4a9) each antibody detects. The HNF4A gene undergoes alternative splicing, generating multiple isoforms that may have different expression patterns and functions. Different antibodies may preferentially detect specific isoforms .

• Cross-Validation with Non-Antibody Methods: Employ techniques that do not rely on antibodies, such as RNA-seq or RT-PCR with isoform-specific primers, to determine HNF4A expression levels independently of antibody-based detection.

• Validation in Knockout/Knockdown Systems: Test all antibody clones in systems where HNF4A has been genetically depleted to confirm specificity. True HNF4A antibodies should show diminished or absent signal in these systems.

• Application-Specific Optimization: Different antibody clones may perform optimally in different applications. For example, an antibody may work well for Western blot but poorly for IHC. Optimize protocols specifically for each antibody and application combination.

• Expanded Target Verification: Use multiple antibodies in parallel to expand coverage of potential targets. As demonstrated in HepG2 ChIP-Seq studies, using multiple antibodies (e.g., combining results with ab41898) can expand the detection of HNF4A-bound gene targets compared to using single antibodies .

• Technical Protocol Reconciliation: Systematically align technical protocols across experiments using different antibodies to minimize method-related differences. This includes standardizing fixation methods, antigen retrieval conditions, blocking protocols, and detection systems.

By implementing these strategies, researchers can better understand the source of contradictory results and develop a more comprehensive and accurate understanding of HNF4A biology in their experimental system.

How can HNF4A antibodies be utilized in studying the role of HNF4A in sepsis-associated lung injury?

Recent research has revealed a protective role for HNF4A in sepsis-associated lung injury, opening new avenues for investigation using HNF4A antibodies:

• Expression Analysis in Disease Models: HNF4A exhibits decreased expression in lung tissues and alveolar macrophages in septic mice (using cecal ligation and puncture method) . Antibodies can be used to quantify this decrease across different sepsis models and severity levels.

• Therapeutic Intervention Monitoring: In experimental models, HNF4A overexpression resulted in increased survival rates and amelioration of pulmonary damage in septic mice . Antibodies can monitor HNF4A expression levels following therapeutic interventions designed to modulate its expression.

• Macrophage Polarization Studies: HNF4A overexpression has been shown to impede M1 polarization while facilitating M2 polarization of macrophages in lung tissues and alveolar lavage fluid . Flow cytometry with HNF4A antibodies combined with M1/M2 markers can assess polarization states at the single-cell level.

• Mechanistic Pathway Analysis: HNF4A transcriptionally regulates nuclear receptor coactivator 2 (NCOA2) through binding to its promoter region. NCOA2 interacts with glucocorticoid receptor (GR) and affects stabilin 1 (STAB1) expression . ChIP experiments using HNF4A antibodies can confirm these binding interactions in relevant cell types.

• Single-Cell Resolution Studies: Using HNF4A antibodies in single-cell techniques like mass cytometry or imaging mass cytometry can identify specific cell populations affected by HNF4A modulation during sepsis.

• Translational Human Studies: Antibodies validated for human samples can be used to investigate HNF4A expression in human sepsis patients, potentially correlating expression levels with disease outcomes and severity.

These approaches utilize HNF4A antibodies to advance understanding of sepsis pathophysiology and potentially identify new therapeutic targets for this condition with high mortality rates .

What methodological considerations are important when using HNF4A antibodies in human pluripotent stem cell differentiation studies?

Human pluripotent stem cell (hPSC) differentiation models are valuable for studying HNF4A in development. When using HNF4A antibodies in these systems, consider these methodological aspects:

• Developmental Timing: HNF4A expression is highly stage-specific during differentiation. Research shows distinct expression patterns in day 14 pancreatic progenitors, day 20 endocrine progenitors, and day 35 beta-like cells for pancreatic differentiation; and in day 8 hepatoblasts for hepatic differentiation . Design experiments to capture these critical developmental windows.

• Antibody Validation: Confirm antibody specificity in stem cell contexts using immunofluorescence, western blot, and flow cytometry analyses, as these systems may express different isoforms or post-translationally modified forms of HNF4A .

• Co-expression Analysis: HNF4A exhibits cross-regulation with other transcription factors like HNF1A. Design multi-parameter detection systems to simultaneously assess these regulatory relationships during differentiation .

• Single-Cell Resolution: Differentiation cultures are heterogeneous. Use flow cytometry or single-cell imaging with HNF4A antibodies to quantify differentiation efficiency and characterize cell populations at the single-cell level.

• Fixation and Permeabilization Optimization: Stem cells and their derivatives may require different fixation protocols than established cell lines. Optimize fixation (PFA concentration and time) and permeabilization conditions specifically for these cellular contexts.

• ChIP-Seq Protocol Adaptation: When studying HNF4A binding patterns during differentiation, adapt ChIP protocols for the smaller cell numbers typically available from differentiation experiments. Micrococcal nuclease (MNase)-based ChIP may provide advantages in these contexts.

• Comparative Analysis with Primary Tissues: Always benchmark antibody staining patterns in differentiated cells against those in primary tissues (liver, pancreas) to assess the fidelity of the in vitro model and antibody performance.

These methodological considerations enable robust investigation of HNF4A's role in differentiation and development, providing insights into both normal development and disease mechanisms .

How can researchers leverage HNF4A antibodies to investigate the transcription factor's role in cancer biology?

While HNF4A is primarily studied in metabolic and developmental contexts, emerging evidence suggests important roles in cancer biology that can be investigated using HNF4A antibodies:

• Expression Profiling Across Cancer Types: Use immunohistochemistry with validated HNF4A antibodies to create a comprehensive expression atlas across cancer types, particularly focusing on cancers of tissues that normally express HNF4A (liver, colon, pancreas, kidney).

• Isoform-Specific Cancer Signatures: Different HNF4A isoforms may have distinct roles in cancer. Use isoform-specific antibodies or antibodies targeting common regions with appropriate controls to distinguish isoform expression patterns in tumor versus normal tissues.

• Epithelial-Mesenchymal Transition (EMT) Studies: As a regulator of epithelial phenotype, HNF4A may influence EMT in cancer. Combine HNF4A antibodies with EMT markers (E-cadherin, vimentin) to assess correlations in cancer progression models.

• Metabolic Reprogramming Analysis: HNF4A regulates numerous metabolic genes. Multiplex immunofluorescence using HNF4A antibodies alongside metabolic enzymes can reveal connections between HNF4A expression and cancer metabolic phenotypes.

• Therapeutic Response Monitoring: Changes in HNF4A expression or localization following cancer treatments can be monitored using validated antibodies, potentially serving as pharmacodynamic biomarkers.

• Cancer Stem Cell Identification: Determine if HNF4A marks cancer stem cell populations by combining HNF4A antibody staining with established cancer stem cell markers in flow cytometry or immunohistochemistry.

• Chromosomal Immunoprecipitation in Cancer Models: Use HNF4A antibodies in ChIP-seq studies to identify cancer-specific changes in HNF4A binding patterns across the genome, revealing altered gene regulation networks.

• Liquid Biopsy Development: Evaluate whether HNF4A protein can be detected in circulation using highly sensitive immunoassays based on validated antibodies, potentially developing minimally invasive cancer biomarkers.

These applications of HNF4A antibodies in cancer research may yield new insights into cancer biology and potentially identify novel therapeutic targets or biomarkers.

What future directions are anticipated for HNF4A antibody development and applications?

The field of HNF4A research is rapidly evolving, with several promising directions for antibody development and applications:

• Isoform-Specific Antibodies: Development of highly specific antibodies that can distinguish between all nine HNF4A isoforms (HNF4a1 through HNF4a9) would enable precise mapping of isoform-specific functions across tissues and disease states.

• Post-Translational Modification (PTM)-Specific Antibodies: Creation of antibodies recognizing specific phosphorylation, acetylation, or other PTM states of HNF4A would provide insights into its regulation and functional modulation in different contexts.

• Therapeutic Antibody Development: Based on findings that HNF4A overexpression mitigates sepsis-associated lung injury , therapeutic antibodies or antibody-drug conjugates targeting HNF4A pathways might be developed for conditions where HNF4A modulation shows benefit.

• Super-Resolution Microscopy-Compatible Antibodies: Engineering of HNF4A antibodies compatible with super-resolution microscopy techniques would enable visualization of HNF4A's nuclear organization and interactions at unprecedented resolution.

• In Vivo Imaging Probes: Development of HNF4A antibody-based PET or SPECT imaging probes could enable non-invasive monitoring of HNF4A expression in disease models and potentially in patients.

• Single-Cell Proteomics Applications: Adaptation of HNF4A antibodies for emerging single-cell proteomic techniques would allow comprehensive mapping of HNF4A expression and its targets at single-cell resolution.

• Antibody Engineering for Tissue Penetration: Engineered antibody formats like single-domain antibodies might improve tissue penetration and signal-to-noise ratio for in vivo applications and thick tissue section staining.

• Multiplexed Detection Systems: Development of compatible antibody panels for simultaneous detection of HNF4A, its binding partners, and downstream targets would enable system-level analysis of HNF4A regulatory networks.