TFAP4 Antibody

What is TFAP4 and why is it a relevant research target?

TFAP4 (Transcription factor AP-4) is a ubiquitously expressed transcription factor that belongs to the basic helix-loop-helix-zipper (bHLH-ZIP) family. It contains a basic domain for DNA binding and HLH and ZIP domains for oligomerization, binding to the symmetrical DNA sequence 5'-CAGCTG-3' to activate both viral and cellular genes . Research interest in TFAP4 has significantly increased due to its upregulation in multiple human malignancies, including hepatocellular carcinoma, colorectal and gastric carcinoma, where it plays crucial oncogenic roles in tumor pathogenesis and progression . Recent studies have also identified TFAP4 as a critical transcriptional regulator in cellular differentiation processes, particularly in bovine satellite cell differentiation , making it a multifaceted research target across diverse biological contexts.

What are the known structural and functional characteristics of TFAP4 protein?

TFAP4 is a protein with a calculated molecular weight of 338 amino acids (approximately 39 kDa), though the observed molecular weight in experimental conditions typically ranges from 39-42 kDa . It is primarily localized in the nucleus where it functions as a transcription factor . Structurally, TFAP4 belongs to the basic helix-loop-helix-zipper (bHLH-ZIP) family and contains a basic domain used for DNA binding and HLH and ZIP domains used for oligomerization . The protein binds to the symmetrical DNA sequence 5'-CAGCTG-3' to activate gene expression . Functionally, TFAP4 has been demonstrated to regulate processes including cellular differentiation, epithelial-mesenchymal transition, and maintenance of progenitor-like states in cancer cells , suggesting its diverse roles in both normal development and disease contexts.

What are the key considerations when selecting a TFAP4 antibody for research?

When selecting a TFAP4 antibody, researchers should consider multiple factors to ensure experimental success. First, identify the specific applications needed (WB, IHC, IF, ChIP, etc.) and select antibodies validated for those applications, as different antibodies show variable performance across techniques . Second, determine appropriate species reactivity—TFAP4 antibodies show reactivity with human, mouse, and rat samples with varying degrees of cross-reactivity . Third, consider clonality options (polyclonal versus monoclonal or recombinant), which affects specificity and batch-to-batch consistency . Fourth, evaluate antibody validation data, including knockout validation when available, which provides the highest confidence in specificity . Finally, review published literature using the specific antibody to assess reliability in conditions similar to your experimental design . Remember that antibody performance may vary depending on sample preparation, fixation methods, and detection systems, so preliminary validation in your specific experimental context is strongly recommended.

What are the optimal conditions for using TFAP4 antibody in Western blotting?

For Western blotting using TFAP4 antibodies, optimal conditions vary by antibody but generally follow this methodological approach: First, prepare total cellular protein by lysing cells with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with a proteinase inhibitor cocktail . Approximately 20 μg of total protein should be separated by SDS-PAGE using 10% separating gel . Transfer proteins to a nitrocellulose membrane through electrophoresis, then block with 5% non-fat milk in TBST at room temperature for 1 hour . For primary antibody incubation, dilutions vary significantly between antibodies: polyclonal antibodies typically require 1:500-1:3000 dilution , while recombinant antibodies may be used at much higher dilutions (1:5000-1:50000) . Incubate with primary antibody at 4°C overnight, followed by washing twice with TBST and incubation with appropriate secondary antibody (typically anti-rabbit or anti-mouse IgG) . TFAP4 protein typically appears as a band at 39-42 kDa . For optimal results, include positive controls from cell lines known to express TFAP4, such as HeLa, Jurkat, A549, or NCI-H1299 cells .

How should immunohistochemistry protocols be optimized for TFAP4 detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for TFAP4 detection requires attention to several methodological details. Firstly, appropriate sample fixation is crucial—formalin-fixed, paraffin-embedded tissues are commonly used with TFAP4 antibodies for IHC-P applications . Antigen retrieval is essential, with heat-induced epitope retrieval in citrate buffer (pH 6.0) typically recommended. Regarding antibody dilutions, they vary significantly between products, with ranges from 1:50-1:200 reported for some antibodies and others requiring optimization within specific ranges indicated by manufacturers . When evaluating TFAP4 staining patterns, look for primarily nuclear localization, as this is the physiological location of this transcription factor . For scoring and quantification, immunoreactive score (IRS) systems have been utilized in published research, with cutoff values of IRS ≥ 4 frequently used to define high TFAP4 expression . To ensure specificity, include appropriate positive controls (such as HCC tissues known to express TFAP4) and negative controls (by omitting primary antibody or using tissues known to lack TFAP4 expression) . For dual staining protocols with other markers, sequential staining procedures with careful consideration of antibody cross-reactivity are recommended.

What are the protocol considerations for using TFAP4 antibodies in chromatin immunoprecipitation (ChIP) assays?

For chromatin immunoprecipitation (ChIP) assays with TFAP4 antibodies, several protocol considerations are critical for success. Begin with appropriate chromatin preparation—approximately 1 million cells should be used per ChIP reaction, with chromatin fragments of appropriate size (200-500 bp) generated through sonication optimization . For immunoprecipitation, use 2-4 μg of TFAP4 antibody incubated with chromatin fragments in a total volume of around 300 μl containing BSA, protease inhibitor cocktail, ChIP buffer, and protein A-coated magnetic beads at 4°C overnight with gentle rocking . When selecting antibodies for ChIP, ensure they are validated specifically for this application, as not all TFAP4 antibodies perform equally in ChIP assays . Published research has demonstrated successful TFAP4 ChIP using commercial antibodies like those from Santa Cruz Biotechnology (sc-377,042) . For qPCR analysis after ChIP, design primers targeting known or predicted TFAP4 binding sites—the consensus sequence 5'-CAGCTG-3' should be considered when designing primers . Include appropriate controls: input DNA (non-immunoprecipitated chromatin), IgG control (non-specific antibody IP), and positive control regions (known TFAP4 targets like DVL1 or LEF1) . For data interpretation, enrichment should be calculated relative to input and normalized to IgG control to account for non-specific binding.

How can TFAP4 antibodies be effectively used in immunofluorescence applications?

Effective use of TFAP4 antibodies in immunofluorescence (IF) requires several methodological considerations. Cells should be fixed with 4% paraformaldehyde for 15-20 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes . Blocking with 1-5% BSA or normal serum in PBS is recommended before antibody incubation. Optimal antibody dilutions vary significantly between products: recombinant TFAP4 antibodies typically require 1:200-1:800 dilution , while other antibodies may require different dilution ranges specified by manufacturers . Incubate primary antibody at 4°C overnight or at room temperature for 1-2 hours, followed by washing with PBS and incubation with fluorophore-conjugated secondary antibodies . When imaging TFAP4 staining, expect primary nuclear localization, which can be confirmed by co-staining with nuclear markers like DAPI . For co-localization studies with other proteins, select secondary antibodies with non-overlapping emission spectra. Validation controls should include samples with known TFAP4 expression (such as HeLa cells) , TFAP4-knockdown samples as negative controls , and secondary-only controls to assess background fluorescence. For quantitative analysis, consistent image acquisition parameters and threshold settings are essential to allow accurate comparison between experimental conditions.

What approaches can be used to study TFAP4's role in cellular differentiation using TFAP4 antibodies?

To study TFAP4's role in cellular differentiation using TFAP4 antibodies, researchers can employ several sophisticated approaches. Temporal expression analysis using Western blotting with TFAP4 antibodies during differentiation time courses can reveal dynamic changes in TFAP4 levels, as demonstrated in studies of bovine satellite cell differentiation . ChIP-seq combining TFAP4 antibodies with next-generation sequencing allows genome-wide identification of TFAP4 binding sites during differentiation processes, helping identify direct transcriptional targets that regulate differentiation . For spatial expression patterns, immunofluorescence with TFAP4 antibodies can be combined with differentiation markers to visualize TFAP4 localization in heterogeneous cell populations, particularly in tissue sections or differentiation cultures . Functional studies combining TFAP4 knockdown or overexpression with differentiation assays, followed by protein analysis using TFAP4 antibodies, have revealed TFAP4's critical role in myofiber differentiation . Co-immunoprecipitation using TFAP4 antibodies can identify differentiation-specific protein interaction partners that modify TFAP4 function during cellular transitions. For epigenetic regulation studies, combine TFAP4 ChIP with histone modification ChIP to understand how TFAP4 affects chromatin states during differentiation. Additionally, proteomic analysis of TFAP4-immunoprecipitated complexes at different differentiation stages can provide comprehensive insights into the molecular mechanisms by which TFAP4 regulates cell fate decisions and lineage commitment.

How can TFAP4 antibodies be used in the context of gene regulatory network analysis?

TFAP4 antibodies enable sophisticated analyses of gene regulatory networks through several methodological approaches. ChIP-seq experiments using TFAP4 antibodies provide genome-wide binding profiles, allowing identification of direct target genes and motif analysis—studies have identified the CAGCTG sequence as the primary TFAP4 binding motif . For integrative network analysis, combine TFAP4 ChIP-seq data with RNA-seq following TFAP4 modulation (verified by Western blotting) to distinguish direct transcriptional effects from secondary consequences, as demonstrated in studies identifying TFAP4 regulation of DVL1 and LEF1 in Wnt signaling . Sequential ChIP (Re-ChIP) using TFAP4 antibodies followed by antibodies against potential co-factors helps identify collaborative transcription factor complexes that co-regulate target genes. Proximity ligation assays using TFAP4 antibodies with antibodies against other transcription factors can visualize protein-protein interactions at regulatory regions in situ. For enhancer analysis, combine TFAP4 ChIP with histone modification ChIP (H3K27ac, H3K4me1) to identify TFAP4-bound active enhancers—research has identified TFAP4 enrichment at active enhancers in differentiating bovine satellite cells . CRISPR-mediated genomic editing of TFAP4 binding sites, followed by antibody-based validation of disrupted binding, provides functional characterization of specific regulatory elements. Additionally, time-course analysis of TFAP4 binding following stimuli can reveal dynamic aspects of regulatory networks, providing comprehensive understanding of how TFAP4 coordinates gene expression programs in various biological contexts.

What are common issues when using TFAP4 antibodies and how can they be resolved?

When working with TFAP4 antibodies, researchers may encounter several common issues requiring systematic troubleshooting. For weak or absent signals in Western blots, first verify TFAP4 expression in your sample—use positive control lysates from A549, HeLa, Jurkat, or NCI-H1299 cells, which are known to express TFAP4 . Optimize protein extraction using RIPA buffer supplemented with protease inhibitors to prevent degradation . If problems persist, try adjusting antibody concentration, extending incubation time, or switching to a more sensitive detection system. For high background in immunohistochemistry or immunofluorescence, implement more stringent blocking protocols with 5% BSA or normal serum from the same species as the secondary antibody . Optimize washing steps and consider titrating both primary and secondary antibodies to find optimal signal-to-noise ratios. When encountering non-specific bands in Western blots, validate antibody specificity using knockout or knockdown controls , and consider using gradient gels to better resolve proteins in the 39-42 kDa range where TFAP4 is detected . For ChIP assays with poor enrichment, optimize chromatin sonication to generate appropriate fragment sizes (200-500 bp), increase antibody amount (up to 4 μg per reaction), and extend incubation times . If cross-reactivity issues occur in multi-color immunofluorescence, select primary antibodies from different host species and use highly cross-adsorbed secondary antibodies to minimize non-specific binding.

How should researchers interpret variations in TFAP4 expression patterns across different samples or conditions?

When interpreting variations in TFAP4 expression patterns, researchers should consider multiple methodological and biological factors. First, establish standardized quantification methods—for Western blots, normalize TFAP4 signals to loading controls like β-tubulin as demonstrated in published research ; for immunohistochemistry, use consistent scoring systems such as the immunoreactive score (IRS) with established cutoffs (e.g., IRS ≥ 4 for high expression) . Consider cell type-specific expression patterns—TFAP4 shows nuclear localization across cell types but expression levels vary, with notably high expression in cancer cells compared to normal cells . When analyzing tissue samples, account for heterogeneity by examining multiple fields and using appropriate statistical methods to quantify differences. For longitudinal studies or treatment responses, establish baseline expression and use consistent detection methods throughout the experimental timeline. Be aware that processing methods can affect antibody epitope accessibility—different fixation protocols may yield varying staining intensities that don't necessarily reflect actual expression differences . When comparing results across different antibodies, recognize that each antibody may detect different epitopes with varying efficiency . For disease-associated expression changes, correlate with clinicopathological parameters and survival data when available, as demonstrated in HCC studies showing TFAP4 overexpression associates with poorer prognosis . Finally, validate key findings using complementary techniques (e.g., combine protein detection with mRNA analysis) to strengthen interpretation of expression pattern variations.

What considerations are important when analyzing TFAP4 binding data from ChIP experiments?

When analyzing TFAP4 binding data from ChIP experiments, several important considerations ensure proper interpretation. First, implement appropriate controls—include input DNA (non-immunoprecipitated chromatin) to normalize for starting material differences, IgG control to account for non-specific binding, and positive control regions targeting known TFAP4 binding sites when available . For peak calling in ChIP-seq analysis, optimize parameters based on TFAP4's known binding characteristics—the consensus CAGCTG motif should be enriched in identified peaks . When interpreting binding intensity variations, consider that binding strength may not directly correlate with transcriptional effect—integrate RNA-seq data to identify functionally relevant binding events . Be aware of cell type-specific binding patterns—TFAP4 may regulate different target genes in different cellular contexts, as seen in its differential roles in cancer progression versus differentiation . For motif analysis, examine both primary TFAP4 binding sites (CAGCTG) and potential co-binding motifs that may indicate collaborative regulation with other transcription factors. Consider chromatin accessibility and histone modification landscapes (e.g., H3K27ac, H3K4me1) when interpreting binding significance, as demonstrated in studies showing TFAP4 enrichment at active enhancers in differentiating cells . For validation of key targets, perform directed ChIP-qPCR with multiple primer sets targeting the binding region. Finally, functional validation through reporter assays or target gene expression analysis following TFAP4 modulation is essential to establish the biological relevance of identified binding events.

How should experiments be designed to study TFAP4's role in different biological contexts?

Designing experiments to study TFAP4's role across biological contexts requires thoughtful methodological approaches. First, implement appropriate model selection—cell lines with endogenous TFAP4 expression (HeLa, A549, etc.) provide accessible systems, while primary cells better represent physiological conditions, as demonstrated in bovine satellite cell studies . For loss-of-function approaches, compare siRNA knockdown (transient, less off-target effects) with CRISPR-Cas9 knockout (complete elimination) approaches, and validate efficiency using TFAP4 antibodies in Western blots . Gain-of-function experiments should use expression plasmids with appropriate promoters, either constitutive or inducible, with careful attention to expression levels relative to endogenous TFAP4 . For pathway analysis, complement TFAP4 modulation with inhibitors of relevant signaling pathways—studies have linked TFAP4 to Wnt/β-catenin and PI3K/AKT pathways . Time-course experiments are essential to distinguish primary from secondary effects, particularly when studying dynamic processes like differentiation or stimulus response . In tissue studies, analyze TFAP4 expression across development stages or disease progression using consistent immunohistochemistry protocols and scoring methods . For transcriptional regulation studies, combine ChIP with reporter assays using wild-type and mutated TFAP4 binding sites . Finally, in vivo approaches using conditional knockout models or xenograft studies with TFAP4-modified cells provide systemic context for cell-based findings , ensuring comprehensive understanding of TFAP4 function across diverse biological settings.

What controls and validation approaches are essential when conducting TFAP4 antibody-based research?

Robust controls and validation approaches are essential for reliable TFAP4 antibody-based research. First, implement specificity controls—ideally, include TFAP4 knockout or knockdown samples to confirm antibody specificity, as demonstrated in studies using siRNA-mediated TFAP4 knockdown . Western blotting validation should confirm a single band at the expected molecular weight (39-42 kDa), with known positive control cell lines (HeLa, A549, Jurkat, NCI-H1299) . For immunohistochemistry or immunofluorescence, include positive control tissues with known TFAP4 expression and negative controls omitting primary antibody . When available, validate findings using multiple TFAP4 antibodies targeting different epitopes to ensure consistent results . For ChIP experiments, include input controls, IgG controls, and positive control regions with established TFAP4 binding . Technical validation should include antibody titration experiments to determine optimal concentrations for each application, avoiding saturation effects that may mask expression differences . Biological validation can be achieved by correlating protein detection with mRNA expression or demonstrating expected biological responses to TFAP4 modulation . For quantitative analyses, implement appropriate statistical methods and blinded scoring when possible to minimize bias. Finally, interlaboratory validation through comparison with published literature strengthens confidence in findings, especially when using established antibody clones with documented performance characteristics , ensuring robust and reproducible TFAP4 research outcomes.

What are the key methodological considerations when studying TFAP4 interactions with other proteins or transcription factors?

When studying TFAP4 interactions with other proteins or transcription factors, several methodological considerations are crucial. For co-immunoprecipitation experiments, use optimized lysis conditions that preserve protein-protein interactions—typically, milder detergent concentrations (0.1-0.5% NP-40) than those used for standard Western blotting, with salt concentrations around 150 mM NaCl . Validate antibody performance in immunoprecipitation applications—not all TFAP4 antibodies that work in Western blotting will efficiently immunoprecipitate the protein . Consider both forward and reverse co-IP approaches, pulling down with TFAP4 antibody and probing for interaction partners, and vice versa . For in situ interaction detection, proximity ligation assays using TFAP4 antibodies paired with antibodies against suspected interaction partners provide spatial information about interactions in their native cellular context. In ChIP-based approaches, sequential ChIP (Re-ChIP) can identify transcription factor co-binding at specific genomic loci—first immunoprecipitate with TFAP4 antibody, then perform a second IP with antibody against potential co-factors . For functional interaction studies, combine co-expression with reporter assays containing TFAP4 binding sites, comparing transcriptional effects of TFAP4 alone versus TFAP4 with interaction partners. When analyzing data, account for context-specific interactions—TFAP4 may interact with different partners in different cell types or under different conditions, as suggested by its varied roles in cancer versus differentiation . Finally, validate key interactions using complementary methods (e.g., combining co-IP with proximity ligation and functional assays) to establish biological relevance of identified interactions.

What are the most promising approaches for studying TFAP4's role in cellular reprogramming and differentiation?

Studying TFAP4's role in cellular reprogramming and differentiation is advancing through several promising methodological approaches. Single-cell analysis combining TFAP4 immunofluorescence with differentiation markers enables tracking of expression dynamics at the individual cell level during fate transitions, providing insights into heterogeneous responses within populations . Genome-wide approaches integrating TFAP4 ChIP-seq with ATAC-seq and RNA-seq allow comprehensive mapping of TFAP4's influence on chromatin accessibility and gene expression programs during differentiation, as demonstrated in satellite cell studies . For mechanistic studies, CRISPR/Cas9-mediated mutagenesis of TFAP4 binding sites at specific enhancers, followed by differentiation assays, can establish causality between TFAP4 binding and differentiation outcomes. Time-course experiments capturing dynamic TFAP4 binding and expression changes during differentiation provide insights into the temporal regulation of cell fate transitions . For pathway integration, combined modulation of TFAP4 with key differentiation signaling pathways helps position TFAP4 within broader regulatory networks—studies have begun connecting TFAP4 to master regulators of differentiation such as MYF5 and MYOG in myogenic contexts . In vivo lineage tracing combined with TFAP4 conditional knockout approaches in model organisms would provide system-level understanding of TFAP4's role in development. Emerging technologies like CUT&RUN or CUT&Tag may offer advantages over traditional ChIP for TFAP4 binding analysis with lower cell input requirements. Additionally, proteomics approaches identifying TFAP4 post-translational modifications during differentiation could reveal regulatory mechanisms controlling its activity across cell fate transitions, advancing our understanding of how this transcription factor coordinates differentiation programs in diverse cellular contexts .