Tead4 Antibody

What is the optimal dilution for TEAD4 antibodies in Western blot applications?

The optimal dilution for TEAD4 antibodies in Western blot applications typically ranges from 1:1000 to 1:5000, though this can vary depending on the specific antibody and sample type. For instance, Proteintech's TEAD4 antibody (12418-1-AP) recommends a dilution range of 1:1000-1:5000 for Western blot . It is strongly recommended to perform a titration experiment to determine the optimal concentration for your specific experimental system. The optimal dilution may be sample-dependent, so validation data should be consulted for different cell lines and tissue types.

What is the expected molecular weight for TEAD4 detection in Western blot?

While the calculated molecular weight of TEAD4 is 48 kDa (based on its 434 amino acid sequence), the observed molecular weight in Western blot typically ranges from 50-55 kDa . This discrepancy between calculated and observed molecular weight is likely due to post-translational modifications. When validating a new TEAD4 antibody, researchers should expect to see bands in this 50-55 kDa range.

Which cell lines show positive TEAD4 expression for antibody validation?

Several cell lines have been validated for TEAD4 expression and can be used as positive controls when testing new antibodies. According to the validation data, the following human cancer cell lines show reliable TEAD4 expression in Western blot:

• BGC-823 cells (gastric cancer)

• HepG2 cells (liver cancer)

• MCF-7 cells (breast cancer)

• MDA-MB-231 cells (breast cancer)

Additionally, colorectal cancer cell lines like HCT116 and SW480 have been used in TEAD4 studies and can serve as positive controls .

What is the subcellular localization of TEAD4 protein?

TEAD4 is primarily a nuclear protein. Immunofluorescence studies have consistently shown nuclear localization of TEAD4 in various cell types. In preimplantation embryos, TEAD4 signals were detected in the nuclei of all blastomeres . While there may be position-dependent differences in signal intensities that become evident from the 32-cell stage onward, the TEAD4 signals remain localized in the nuclei . In knockout validation studies, the nuclear signals disappeared while weak cytoplasmic signals remained, demonstrating the specificity of the nuclear staining .

What species reactivity can be expected from TEAD4 antibodies?

TEAD4 antibodies show varying species reactivity depending on the product. Many commercially available antibodies react with human, mouse, and rat TEAD4 . Some antibodies have broader reactivity, including guinea pig, horse, rabbit, cow, dog, and even zebrafish . When selecting an antibody, it's important to verify the reactivity against your species of interest. This table summarizes the reactivity of a typical TEAD4 antibody:

What applications are TEAD4 antibodies typically validated for?

TEAD4 antibodies have been validated for multiple applications in molecular and cellular biology research. According to the search results, commonly validated applications include:

The selection of application should guide your choice of antibody, as not all antibodies perform equally well across all applications .

How can I validate the specificity of a TEAD4 antibody?

Validating antibody specificity is crucial for reliable results. For TEAD4 antibodies, several approaches are recommended:

• Knockdown/Knockout validation: Perform Western blot or immunostaining using samples from TEAD4 knockdown or knockout models. In TEAD4 knockout embryos, specific nuclear signals disappeared while weak cytoplasmic signals remained, confirming antibody specificity .

• Multiple antibody comparison: Use antibodies targeting different epitopes of TEAD4 and compare the staining patterns.

• Molecular weight verification: In Western blot, verify that the detected band aligns with the expected molecular weight (50-55 kDa) .

• Positive and negative controls: Include cell lines known to express TEAD4 (e.g., BGC-823, HepG2, MCF-7) as positive controls .

• Peptide blocking: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity.

How does TEAD4 function in the Hippo signaling pathway?

TEAD4 performs an essential role in the Hippo signaling pathway. It regulates gene expression of YAP1 and WWTR1/TAZ, thus mediating cell proliferation, migration, and epithelial-mesenchymal transition induction . The transcriptional activity of TEAD4 requires several regions of the protein, a TBP-associated factor, and limiting transcriptional intermediary factors .

Interestingly, while TEAD4 typically works with YAP/TAZ in the Hippo pathway, research has shown that in nasopharyngeal carcinoma (NPC), TEAD4 can promote cancer progression independently of YAP/TAZ modulation . This suggests context-specific functions of TEAD4 that may vary between different cell types and cancer models.

When designing experiments to study TEAD4's role in the Hippo pathway, researchers should consider using co-immunoprecipitation assays to detect TEAD4-YAP/TAZ interactions and ChIP-seq to identify direct targets of TEAD4 .

What cancer types have been associated with TEAD4 expression?

TEAD4 has been implicated in several cancer types, with evidence suggesting it plays a tumor-promoting role. According to the search results:

• Colorectal cancer: TEAD4 plays an important tumor-promoting role by directly targeting YAP1. Knockdown of TEAD4 suppressed tumorigenesis in vivo .

• Urinary bladder cancer (UBC): TEAD4 is strikingly elevated in UBC tissues compared to normal counterparts. Upregulation of TEAD4 significantly correlates with clinical stage, pathological grade, and poor clinical outcomes .

• Nasopharyngeal carcinoma (NPC): TEAD4 has been identified as a master regulator of high-risk NPC and can act as a potential therapeutic vulnerability for patients .

These findings suggest that TEAD4 may serve as a biomarker and potential therapeutic target in various cancer types.

What considerations are important when selecting TEAD4 antibodies for different applications?

When selecting TEAD4 antibodies for research, consider these factors:

• Application compatibility: Ensure the antibody is validated for your specific application (WB, IF, IHC, ChIP, etc.) .

• Host species: Consider the host species (rabbit, mouse) in relation to your experimental design, especially for multi-color staining protocols .

• Clonality: Monoclonal antibodies typically offer higher specificity but narrower epitope recognition, while polyclonal antibodies provide broader epitope recognition but potentially more background .

• Epitope region: Antibodies targeting different regions (N-terminal, C-terminal, middle region) may perform differently depending on protein interactions or modifications .

• Validation data: Review published studies that have used the antibody successfully in your application of interest .

What methodological approaches can address contradictory results in TEAD4 subcellular localization studies?

Contradictory results regarding TEAD4 subcellular localization have been reported in the literature. For instance, Home et al. proposed a model where regulated nuclear localization of TEAD4 controls trophectoderm vs. inner cell mass formation, while Nishioka et al. suggested a model where TEAD4 is constitutively nuclear . To address such contradictions:

• Antibody validation: Thoroughly validate antibody specificity using knockout/knockdown controls. In the cited study, researchers repeated immunostaining experiments using the same antibodies as used in the contradictory study .

• Multiple antibody approach: Use several antibodies targeting different epitopes of TEAD4 to confirm localization patterns.

• Complementary techniques: Combine immunofluorescence with biochemical fractionation to quantitatively assess nuclear vs. cytoplasmic distribution.

• Tagged protein expression: Use epitope-tagged TEAD4 expression to confirm antibody staining patterns.

• Context consideration: Document the exact experimental conditions, as localization may be cell-type, developmental stage, or context-dependent.

In the study addressing contradictory results, researchers found that Tead4 proteins are present in the nuclei of all blastomeres throughout preimplantation development, consistent with one model but differing from the results of another study .

How can ChIP-seq be optimized for studying TEAD4 binding sites?

ChIP-seq is a powerful technique for identifying genome-wide binding sites of TEAD4. Based on published methodologies:

• Antibody selection: Use ChIP-validated TEAD4 antibodies. In the cited study, immunoprecipitated chromatin fragments were used to prepare libraries for deep sequencing .

• Controls: Include appropriate controls such as total chromatin fragments before immunoprecipitation and immunoprecipitated chromatin fragments using a nonspecific antibody (e.g., mouse IgG) .

• Peak calling: Use established algorithms like Model-based Analysis of ChIP-Seq to identify specific targets of TEAD4 with high significance values (e.g., 1 × 10^-5) .

• Motif analysis: Apply overrepresented motif detection algorithms like Hypergeometric Optimization of Motif Enrichment (HOMER) to identify motifs localized within TEAD4 binding regions .

• Integration with gene expression data: Correlate binding sites with gene expression changes to identify direct functional targets.

In a study of mouse trophoblast stem cells, researchers identified 4,949 TEAD4 binding regions and found that the TEAD motif was the most prevalent motif at these binding regions with a P value of 1 × 10^-89 .

How can I design experiments to investigate TEAD4's role in cancer progression?

Based on the search results, a comprehensive experimental design to investigate TEAD4's role in cancer might include:

• Expression analysis: Compare TEAD4 expression between tumor and normal tissues using techniques like RT-PCR, Western blot, and immunohistochemistry. In colorectal adenoma studies, TEAD4 expression was significantly higher in CRA tissues than in their normal counterparts .

• Clinical correlation: Analyze the relationship between TEAD4 expression and clinicopathological features. In UBC, TEAD4 protein level was remarkably correlated with T stage (P=0.002) and tumor grade (P<0.001) .

• Functional studies: Perform knockdown or overexpression of TEAD4 to assess effects on cell proliferation, migration, and invasion. In colorectal cancer, knockdown of TEAD4 markedly suppressed tumor growth in vivo .

• Mechanistic investigations: Use techniques like RNA-seq, ChIP-seq, and luciferase reporter assays to identify downstream targets and signaling pathways. In colorectal cancer, TEAD4 regulated YAP1 by direct binding and transcriptional activation .

• In vivo models: Establish xenograft tumor models to validate in vitro findings. HCT116 cells with TEAD4 shRNA showed significantly smaller tumors compared to control shRNA .

What approaches can be used to study TEAD4's YAP/TAZ-independent functions?

Recent research has revealed that TEAD4 can function independently of YAP/TAZ in certain contexts. To study these independent functions:

• Protein interaction analysis: Perform co-immunoprecipitation assays to assess the endogenous interaction between TEAD4 and YAP/TAZ. In NPC cells, this interaction was found to be weak compared to other cancer types .

• Gel filtration chromatography: Analyze protein complexes to determine if TEAD4 forms complexes with YAP/TAZ. In NPC, low levels of TEAD4 formed complexes with YAP/TAZ .

• Mutant studies: Construct plasmids encoding YAP/TAZ binding–deficient TEAD4 mutants (e.g., TEAD4 Y429H, TEAD4 K297A, and TEAD4 W299A) to study YAP/TAZ-independent functions .

• Rescue experiments: In TEAD4-knockdown cells, restore the expression of both wild-type TEAD4 and YAP/TAZ binding–deficient mutants to compare their ability to rescue phenotypes .

• YAP/TAZ knockout: Create YAP/TAZ double-knockout cells and assess the effects of TEAD4 expression on cellular phenotypes .

In NPC studies, restoring expression of YAP/TAZ binding–deficient TEAD4 mutants efficiently rescued impaired migration, invasion, and cisplatin resistance in TEAD4-knockdown cells, indicating YAP/TAZ-independent functions .

How should target validation be performed after identifying potential TEAD4-regulated genes?

After identifying potential TEAD4 target genes through methods like ChIP-seq or expression profiling, rigorous validation is necessary:

• Expression correlation: Analyze the correlation between TEAD4 and potential target gene expression. In NPC studies, researchers identified genes showing significant correlation (R > 0.4 and P < 0.05) with TEAD4 levels .

• Quantitative RT-PCR: Verify expression changes of potential targets after TEAD4 overexpression or knockdown. This approach identified BZW2, LARP6, and RCSD1 as significantly regulated by TEAD4 .

• Western blot: Confirm protein-level changes of potential targets following TEAD4 manipulation .

• ChIP-qPCR: Use chromatin immunoprecipitation followed by qPCR to confirm direct binding of TEAD4 to promoter regions of target genes. In colorectal cancer studies, ChIP-qPCR with anti-TEAD4 antibody revealed significant enrichment of TEAD4 binding in the proximal region containing TEAD4-binding motif .

• Luciferase reporter assay: Design reporters containing the target gene promoter region with wild-type or mutated TEAD4-binding motifs to confirm direct transcriptional regulation .

These approaches have successfully validated targets such as YAP1 in colorectal cancer and BZW2 in nasopharyngeal carcinoma .

What are the optimal immunohistochemistry protocols for TEAD4 detection in FFPE samples?

For optimal TEAD4 detection in formalin-fixed paraffin-embedded (FFPE) samples:

• Sample preparation: Deparaffinize and rehydrate sections according to standard protocols .

• Antigen retrieval: Microwave and boil sections in 10 mM citrate buffer (pH 6.0) .

• Endogenous peroxidase blocking: Incubate sections in 0.3% hydrogen peroxide for approximately 15 minutes .

• Nonspecific binding blocking: Block nonspecific antigens using serum (e.g., sheep serum) for 30 minutes .

• Primary antibody incubation: Incubate with TEAD4 antibody at an optimized dilution (typically 1:200) overnight at 4°C .

• Secondary antibody and detection: Follow standard streptavidin-biotin-peroxidase complex method procedures .

• Controls: Include sections incubated with PBS instead of primary antibody as negative controls .

For immunoperoxidase staining with monoclonal antibodies to TEAD4 on FFPE human placenta, an antibody concentration of 3 μg/ml has been reported to be effective .

What strategies can optimize Co-IP experiments to detect TEAD4 protein interactions?

Co-immunoprecipitation (Co-IP) is crucial for studying TEAD4 protein interactions. To optimize Co-IP experiments:

• Antibody selection: Choose Co-IP validated TEAD4 antibodies. Some antibodies have been specifically validated for Co-IP applications .

• Lysis conditions: Use appropriate lysis buffers that preserve protein-protein interactions while effectively extracting nuclear proteins like TEAD4.

• Crosslinking consideration: For transient or weak interactions, consider using reversible crosslinking reagents.

• Reciprocal Co-IP: Perform Co-IP in both directions (e.g., immunoprecipitate with anti-TEAD4 and detect YAP/TAZ, then immunoprecipitate with anti-YAP/TAZ and detect TEAD4).

• Controls: Include appropriate controls such as IgG control immunoprecipitation and input samples.

When studying TEAD4-YAP/TAZ interactions, researchers found that the endogenous interaction between TEAD4 and YAP/TAZ was weak in NPC cells compared with breast cancer or bladder cancer cells . This highlights the importance of considering cell type-specific differences in protein interactions.

How can TEAD4 ChIP experiments be optimized for different cell types?

Optimizing TEAD4 ChIP experiments requires consideration of several factors:

• Crosslinking conditions: Optimize formaldehyde concentration (typically 1%) and crosslinking time (typically 10-15 minutes) for nuclear transcription factors like TEAD4.

• Chromatin fragmentation: Ensure optimal sonication conditions to generate DNA fragments of 200-500 bp.

• Antibody selection: Use ChIP-validated TEAD4 antibodies. Several commercial antibodies have been validated for ChIP applications .

• Antibody amount: Optimize the amount of antibody used for immunoprecipitation, typically 2-5 μg per reaction.

• Controls: Include appropriate controls such as IgG immunoprecipitation and input chromatin.

• Cell-type considerations: For cell types with lower TEAD4 expression, increase cell numbers and optimize lysis conditions.

• qPCR primer design: Design qPCR primers targeting known or predicted TEAD4 binding sites based on consensus motifs.

In one study, researchers performed ChIP-qPCR with anti-TEAD4 antibody to assess TEAD4 binding to the YAP1 promoter region in HCT116 and SW480 cells. Results revealed significant enrichment of TEAD4 binding in the proximal region containing the TEAD4-binding motif .