TFAP2E Antibody

What is TFAP2E and why is it important in research?

TFAP2E (transcription factor AP-2 epsilon) is a member of the AP-2 family of transcription factors with a calculated molecular weight of 46 kDa (442 amino acids). It functions as an activating enhancer binding protein and plays significant roles in neural development and gene regulation. TFAP2E is particularly important in neurodevelopmental research as it is expressed in maturing cells fated to become V2R neurons and controls parts of the basal and apical VSN (vomeronasal sensory neuron) genetic programs. Recent studies have combined scRNA-seq, histology, behavior, and CUT&RUN techniques to demonstrate TFAP2E's involvement in sociosexual behavior regulation . Additionally, in cancer research, TFAP2E methylation status has been studied as a potential predictor of response to 5-fluorouracil-based chemotherapy in colorectal cancer patients .

What applications are TFAP2E antibodies most commonly used for?

TFAP2E antibodies are primarily employed in the following experimental applications:

These applications allow researchers to investigate TFAP2E expression, localization, and function in various biological contexts . The antibody has demonstrated reactivity with human, mouse, and rat samples, making it versatile for comparative studies across species .

How should I determine the appropriate dilution for my specific experiment?

While standard dilution ranges are provided (as shown in the table above), the optimal dilution for TFAP2E antibody can vary significantly depending on your experimental conditions. It is recommended that researchers titrate the antibody in each testing system to obtain optimal results. Sample-dependent variations are common, so preliminary experiments with a dilution series are advised. For example, in immunohistochemistry applications with human skin cancer tissue, a 1:200 dilution has been successfully used with heat-mediated antigen retrieval using Tris-EDTA buffer (pH 9.0) . For Western blot applications with A375 cells, a 1:600 dilution incubated at room temperature for 1.5 hours has shown good results .

What is the recommended protocol for antigen retrieval when using TFAP2E antibodies in IHC?

For optimal antigen retrieval when using TFAP2E antibodies in immunohistochemistry applications, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) is the primary recommended method. This has been successfully demonstrated in applications such as human skin cancer tissue staining . Alternatively, if issues arise with this method, antigen retrieval may also be performed with citrate buffer at pH 6.0, though this is considered a secondary option .

The effectiveness of antigen retrieval can significantly impact staining quality, as TFAP2E is a nuclear transcription factor that may require stronger retrieval conditions to expose epitopes that are masked during fixation processes. When evaluating TFAP2E expression by IHC, proper antigen retrieval ensures accurate nuclear localization of the staining pattern, which is crucial for distinguishing between positive and negative expression.

How can I distinguish between specific and non-specific staining with TFAP2E antibodies?

Distinguishing between specific and non-specific staining when using TFAP2E antibodies requires careful experimental design and proper controls. According to established scoring criteria:

• A tumor should be considered to have normal expression for TFAP2E when unequivocal nuclear staining is observed in the neoplastic epithelial cells, with or without cytoplasmic staining .

• Staining intensity can be scored as follows:

  • Score 3: Strong and homogeneous staining at 10X magnification

  • Score 2: Strong but heterogeneous staining at 20X magnification

  • Score 1: Light and heterogeneous staining at 40X magnification

• Tumor cells should be judged as negative for TFAP2E expression only if they lack nuclear staining in a sample where stromal cells show positive staining (serving as internal positive controls) .

• Samples should not be scored when no staining of internal control is visible, as this suggests technical issues rather than true negative expression .

Including appropriate positive and negative controls in each experiment is essential. The use of tissues known to express TFAP2E (such as brain tissue for endogenous expression) alongside your experimental samples can help validate staining patterns.

What are the most effective approaches for validating TFAP2E antibody specificity?

Validating the specificity of TFAP2E antibodies requires a multi-faceted approach:

• Genetic models: Using tissues from TFAP2E knockout models provides the gold standard for antibody validation. Research has shown that in Null Tfap2e mice, where Cre was knocked into the DNA binding domain, only faint cytoplasmic immunoreactivity limited to marginal zones was observed, while rescue Tfap2e mice showed restored AP-2ε immunoreactivity in the basal region of the vomeronasal epithelium (VNE) . These genetic controls confirm antibody specificity.

• Western blot analysis: Confirming that the antibody detects a protein of the expected molecular weight (46 kDa for TFAP2E) in positive control samples (such as A375 cells) while showing no bands in negative control samples .

• Peptide competition assays: Pre-incubating the antibody with excess recombinant TFAP2E protein or immunizing peptide should abolish specific staining in immunohistochemistry or Western blot applications.

• Correlation with mRNA expression: Comparing antibody staining patterns with mRNA expression data from in situ hybridization or RNA-seq can provide additional validation of specificity.

• Cross-reactivity testing: Testing the antibody against related proteins (other TFAP2 family members) to ensure it doesn't cross-react with similar epitopes.

How can TFAP2E antibodies be used to study transcriptional regulatory networks?

TFAP2E antibodies can be powerful tools for studying transcriptional regulatory networks through several advanced approaches:

• Chromatin Immunoprecipitation (ChIP) followed by sequencing: CUT&RUN (Cleavage Under Targets and Release Using Nuclease) with TFAP2E antibodies has been used to determine direct genetic targets of AP-2ε in the vomeronasal organ (VNO). This approach identified over 5,025 replicable peaks indicating AP-2ε binding sites in VNO tissue. After filtering against peaks from TFAP2E knockout controls, 4,871 genuine binding sites remained, which were assigned to 3,186 genes . This technique allows researchers to map the genome-wide occupancy of TFAP2E and identify its direct target genes.

• Co-immunoprecipitation (Co-IP): TFAP2E antibodies can be used to pull down TFAP2E along with its interacting protein partners, allowing for the identification of transcriptional complexes and co-factors that work with TFAP2E to regulate gene expression.

• Sequential ChIP (ChIP-reChIP): This technique involves performing ChIP with a TFAP2E antibody followed by a second ChIP with antibodies against other transcription factors to identify genomic regions where TFAP2E works in concert with specific partners.

• Integration with transcriptomic data: Combining ChIP-seq data with RNA-seq from TFAP2E knockout or overexpression models helps establish direct relationships between TFAP2E binding and gene expression changes, revealing its activating or repressive functions in different contexts.

What is the relationship between TFAP2E methylation and protein expression, and how can antibodies help investigate this?

The relationship between TFAP2E methylation and protein expression is complex and has significant implications for disease progression and treatment response, particularly in colorectal cancer:

How can TFAP2E antibodies be used to investigate neuronal development and function?

TFAP2E antibodies are valuable tools for investigating neuronal development and function, particularly in specialized neural systems:

• Neuronal lineage tracing: TFAP2E is expressed in maturing cells fated to become V2R neurons. Antibodies against TFAP2E can help track the differentiation and maturation of these neuronal populations during development .

• Functional domain mapping: Studies have revealed that TFAP2E controls parts of both basal and apical vomeronasal sensory neuron (VSN) genetic programs. Using TFAP2E antibodies in combination with other markers can help delineate the organization of functional domains within neural structures .

• Analysis of neural circuit formation: The expression of axon guidance molecules like Robo2 and Nrp2, as well as adhesion molecules Kirrel2 and Kirrel3, has been studied in relation to TFAP2E function. Immunostaining against these molecules in wild-type versus TFAP2E knockout or rescue models helps understand how TFAP2E influences circuit formation and connectivity .

• Behavioral phenotype correlation: TFAP2E has been implicated in the regulation of sociosexual behavior. Correlating TFAP2E expression patterns (detected via antibodies) with behavioral phenotypes can provide insights into the molecular basis of complex behaviors .

Why might TFAP2E show variable subcellular localization in different experimental contexts?

• Developmental stage-dependent localization: In Null Tfap2e mice, faint AP-2ε cytoplasmic immunoreactivity was observed limited to the most marginal zones of the vomeronasal organ (VNO), while no immunoreactivity was detected in the rest of the neuroepithelium . This suggests that subcellular localization may change during developmental progression.

• Rescue versus wild-type expression patterns: In Rescue Tfap2e mice, the AP-2ε expression pattern and immunoreactivity were not identical to controls in neurogenic regions, with no AP-2ε immunoreactivity observed at the tips of the neurogenic niche in the vomeronasal epithelium (VNE) . This indicates that normal regulatory mechanisms may influence proper localization.

• Scoring criteria for research purposes: When evaluating TFAP2E expression by immunohistochemistry, a tumor is considered to have normal expression when unequivocal nuclear staining is observed in neoplastic epithelial cells, with or without cytoplasmic staining . This recognizes that some cytoplasmic staining may occur alongside the expected nuclear localization.

• Technical considerations: Fixation conditions, antigen retrieval methods, and antibody penetration can all affect the apparent subcellular localization of TFAP2E. Overfixation may mask nuclear epitopes, while insufficient permeabilization may prevent antibody access to nuclear antigens.

What are common technical challenges when working with TFAP2E antibodies, and how can they be addressed?

Researchers working with TFAP2E antibodies may encounter several technical challenges:

• Optimizing antigen retrieval: For IHC applications, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) is recommended, but alternative methods using citrate buffer (pH 6.0) may be necessary depending on the tissue type and fixation method . Systematic testing of different antigen retrieval conditions may be required for optimal results.

• Antibody dilution optimization: While recommended dilutions are provided (WB: 1:500-1:1000, IHC: 1:50-1:500, IF/ICC: 1:50-1:500), the optimal dilution can be sample-dependent . Titration experiments should be performed to determine the best antibody concentration for each specific application and sample type.

• Cross-reactivity considerations: When selecting a TFAP2E antibody, it's important to consider potential cross-reactivity with other TFAP2 family members. Different antibodies may recognize different epitopes, affecting specificity. The immunogen information (e.g., TFAP2E fusion protein Ag23023) can help assess potential cross-reactivity .

• Storage and handling: TFAP2E antibodies should be stored according to manufacturer recommendations (e.g., -20°C with 0.02% sodium azide and 50% glycerol at pH 7.3) to maintain stability and activity . Proper aliquoting and handling practices can prevent freeze-thaw cycles that degrade antibody performance.

• Validation in each experimental system: Even well-characterized antibodies should be validated in each new experimental system. This includes confirming the expected molecular weight in Western blot (46 kDa for TFAP2E) and the expected localization pattern in immunostaining applications .

How can discrepancies between TFAP2E protein expression and gene methylation data be reconciled?

Researchers investigating TFAP2E may encounter situations where protein expression data (from antibody-based methods) doesn't align perfectly with gene methylation data. Reconciling these discrepancies requires consideration of several factors:

• Methylation site specificity: TFAP2E contains multiple CpG islands that can be differentially methylated, including regions in the promoter/exon 1 and intron 3 . Different methylation patterns at these distinct sites may have varying impacts on protein expression, requiring comprehensive methylation analysis across all relevant regions.

• Quantitative threshold determination: Studies have used matched tumor and adjacent normal tissue samples to establish methylation thresholds through Receiver Operating Characteristics (ROC) analysis . This statistical approach helps define biologically relevant methylation levels that impact expression, rather than using arbitrary cutoffs.

• Post-transcriptional and post-translational regulation: Protein expression is influenced by factors beyond promoter methylation, including mRNA stability, translational efficiency, and protein degradation. These mechanisms may explain cases where protein expression doesn't directly correlate with methylation status.

• Technical considerations in protein detection: The sensitivity and specificity of antibody-based detection methods can affect apparent protein expression levels. For example, IHC scoring systems that categorize staining intensity (scores 1-3) provide semi-quantitative rather than absolute measures of protein levels .

• Heterogeneity within samples: Cellular heterogeneity within tissue samples can lead to apparently discordant results between bulk methylation analysis and protein expression studies. Single-cell approaches or microdissection techniques may help resolve these discrepancies.

How are TFAP2E antibodies being used to study cancer biomarkers and treatment response?

TFAP2E antibodies are becoming increasingly important in cancer research, particularly in studying biomarkers for treatment response:

• Methylation-expression correlations: TFAP2E methylation status has been investigated as a potential predictor of response to 5-fluorouracil (5FU)-based chemotherapy in colorectal cancer. Antibody-based detection of TFAP2E protein expression provides crucial complementary data to methylation studies, allowing researchers to confirm that epigenetic changes translate to altered protein expression .

• Scoring systems for clinical application: Standardized scoring systems have been developed for evaluating TFAP2E expression by immunohistochemistry in cancer tissues. These systems categorize staining intensity on a scale from 1-3, providing semi-quantitative measures that can be correlated with clinical outcomes .

• Validation in large patient cohorts: The clinical utility of TFAP2E as a biomarker requires validation in large, well-characterized patient cohorts. Studies have examined TFAP2E methylation and expression in cohorts of hundreds of colorectal cancer patients (e.g., 532 from a population-based multicenter cohort and 251 from a clinic-based trial) to assess its predictive value for treatment response .

• Downstream pathway analysis: TFAP2E's effects on treatment response may be mediated by downstream effectors like DKK4. Combined analysis of TFAP2E and its target genes using antibody-based methods helps elucidate the molecular mechanisms underlying treatment resistance .

What novel approaches combine TFAP2E antibodies with other molecular techniques for comprehensive functional studies?

Cutting-edge research is integrating TFAP2E antibody-based methods with other molecular techniques to gain comprehensive insights into its function:

• CUT&RUN with single-cell RNA sequencing: Combining cleavage under targets and release using nuclease (CUT&RUN) with TFAP2E antibodies alongside single-cell RNA sequencing has allowed researchers to map TFAP2E binding sites genome-wide and correlate these with cell type-specific gene expression patterns. This approach identified over 5,025 replicable peaks indicating AP-2ε binding sites, which were assigned to 3,186 genes .

• Integrated epigenomic and transcriptomic analysis: Researchers are integrating data from TFAP2E CUT&RUN experiments with other epigenomic profiling methods (such as ATAC-seq for chromatin accessibility) and transcriptome analysis to build comprehensive models of how TFAP2E regulates gene expression in different cellular contexts.

• Rescue experiments in genetic models: Studies have used genetic models (Null Tfap2e and Rescue Tfap2e mice) in combination with antibody-based detection methods to investigate how restoration of TFAP2E expression affects downstream molecular pathways and phenotypes . This approach helps establish causal relationships between TFAP2E expression and biological functions.

• Multi-parameter imaging: Advanced imaging techniques that allow simultaneous detection of multiple proteins are being used to study how TFAP2E interacts with other factors in intact tissues. These approaches provide spatial information about TFAP2E expression and function that complements biochemical methods.

How does TFAP2E function compare across different species, and what implications does this have for antibody selection?

Understanding cross-species conservation and variation in TFAP2E function has important implications for research and antibody selection:

• Cross-species reactivity: TFAP2E antibodies like 25829-1-AP have demonstrated reactivity with human, mouse, and rat samples . This cross-reactivity reflects the evolutionary conservation of TFAP2E protein structure across mammalian species and allows for comparative studies.

• Validation in different species: When using TFAP2E antibodies across species, validation in each target species is essential. Positive controls for Western blot applications include A375 cells (human), mouse brain tissue, and rat brain tissue , confirming the antibody's utility in multiple species.

• Functional conservation analysis: Studies in mouse models have provided insights into TFAP2E's role in neuronal development and function, particularly in the vomeronasal system and sociosexual behavior regulation . The conservation of these functions in humans remains an area of ongoing investigation, requiring careful validation of antibody-based findings across species.

• Species-specific technical considerations: Different species may require adjustments to experimental protocols when using TFAP2E antibodies. For example, antigen retrieval conditions, antibody dilutions, and incubation times may need optimization for each species to account for differences in tissue composition and fixation properties.

• Recombinant protein availability: For validation and control experiments, recombinant TFAP2E proteins are available from various expression systems (E. coli, Wheat germ, HEK-293 cells) and multiple species origins (human, mouse) . These resources facilitate species-specific validation of antibody performance.