TEAD3 Antibody

What applications are TEAD3 antibodies validated for in research?

TEAD3 antibodies are validated for multiple applications with specific optimal dilutions:

For optimal results, each antibody should be titrated in the specific experimental system as sensitivity can vary between sample types and protocols .

How do I select the appropriate TEAD3 antibody for my research?

Selection criteria should include:

• Target region specificity: Antibodies targeting different regions (N-terminal, middle region, C-terminal) are available . The middle region antibody (e.g., ABIN2777937) shows high cross-species reactivity .

• Clonality considerations:

  • Monoclonal antibodies (e.g., EPR27092-16) offer high specificity and reproducibility with defined epitopes .

  • Polyclonal antibodies provide broader epitope recognition but potential batch-to-batch variability .

• Species reactivity: Available antibodies show reactivity with various species:

  • Human and mouse (most common)

  • Extended reactivity including rat, dog, cow, pig, guinea pig, and zebrafish (for certain products)

• Validation data: Review validation images provided by manufacturers showing proper molecular weight detection (typically ~49-50 kDa) and specific staining patterns .

What storage conditions are recommended for TEAD3 antibodies?

Most TEAD3 antibodies require:

• Long-term storage at -20°C (stable for one year after shipment)

• Short-term/frequent use storage at 4°C (up to one month)

• Storage in buffer containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Avoiding repeated freeze-thaw cycles

Notably, aliquoting is generally unnecessary for -20°C storage according to manufacturer recommendations . Some products (typically 20μL sizes) contain 0.1% BSA for additional stability .

How can I validate TEAD3 antibody specificity for distinguishing between TEAD family members?

TEAD family members (TEAD1-4) share significant sequence homology, requiring careful validation:

• Dot blot analysis: Using recombinant TEAD proteins to confirm reactivity against the intended target. For example, ab309536 demonstrated specific binding to TEAD3 recombinant fragment with minimal cross-reactivity to human TEAD1, TEAD2, and TEAD4 recombinant proteins .

• Western blot validation:

  • TEAD3 antibodies should detect a band at approximately 49-50 kDa .

  • An additional lower molecular weight band (~30 kDa) may represent a TEAD3 splice variant .

  • Positive control lysates include HCT116, HepG2, and MCF-7 cells .

  • Comparative analysis with other TEAD family antibodies can confirm specificity.

• Cellular expression pattern:

  • TEAD3 has strong mRNA expression in placenta and choriocarcinoma cells (JEG-3) .

  • Expression is lower in HeLa, HepG2, and MCF-7 compared to placental tissues .

  • Human pancreas and liver show relatively low expression levels .

What approaches can optimize TEAD3 detection in immunohistochemistry?

Several technical considerations improve IHC detection of TEAD3:

• Antigen retrieval methods:

  • Primary recommendation: TE buffer (pH 9.0)

  • Alternative approach: Citrate buffer (pH 6.0)

• Blocking and dilution conditions:

  • Optimal blocking: 10% normal goat serum for 30 minutes at room temperature

  • Antibody dilution in 1% BSA

  • Primary antibody incubation at 4°C overnight

• Detection systems:

  • Biotinylated secondary antibody followed by HRP-conjugated SP system visualization

  • For fluorescence applications: Secondary antibodies such as Cy3 Goat Anti-Rabbit IgG at 1:500 dilution

• Tissue-specific considerations:

  • Mouse small intestine tissue has been validated for positive IHC detection

  • Human pancreatic cancer tissue has shown strong TEAD3 expression

  • Statistical analysis revealed that TEAD3 mRNA levels were significantly upregulated in human hepatoblastomas compared to matching non-neoplastic tissues

How do TEAD3 antibodies perform in studies examining Hippo pathway activation?

TEAD3 antibodies have been instrumental in elucidating Hippo pathway regulation:

• YAP-TEAD interaction studies:

  • TEAD3 antibodies can be used in co-immunoprecipitation to assess YAP-TEAD3 complex formation .

  • Analysis of TEAD3 thermal stability through CETSA (Cellular Thermal Shift Assay) using antibodies can demonstrate TEAD3 engagement by small molecule inhibitors .

• Transcriptional activity assessment:

  • Antibodies can be used with reporter assays to monitor TEAD-dependent transcription, such as the 8xGTIIC-luciferase reporter system .

  • Immunoprecipitation with TEAD3 antibodies followed by ChIP-seq can identify TEAD3 binding sites at promoters of Hippo pathway target genes .

• Inhibitor validation:

  • TEAD3 antibodies can confirm target engagement of selective inhibitors like DC-TEAD3in03 (IC₅₀ value of 0.16 ± 0.03 μmol/L) .

  • Western blotting with TEAD3 antibodies can verify selective inhibition of TEAD3 versus other TEAD family members after compound treatment .

How are TEAD3 antibodies used in cancer research applications?

TEAD3 antibodies have revealed important roles in cancer biology:

• Expression analysis in tumors:

  • IHC studies using TEAD3 antibodies have identified upregulation in pancreatic cancer .

  • Hepatoblastomas show significantly increased TEAD3 mRNA levels compared to non-neoplastic tissues .

• Therapeutic target assessment:

  • TEAD3 antibodies help evaluate the efficacy of covalent TEAD inhibitors like MYF-03-69, which blocks palmitoylation of all four TEAD paralogs .

  • Covalent inhibitors like DC-TEAD3in03 with 100-fold selectivity over other TEAD isoforms can be validated using TEAD3-specific antibodies .

• Functional studies:

  • Researchers have used TEAD3 antibodies to establish that TEAD4 (not TEAD3) is the major mediator of YAP-associated oncogenesis in certain contexts, highlighting the importance of isoform-specific detection .

  • TEAD2VP16 and ΔN90-β-catenin co-expression resulted in massive liver tumor growth, demonstrating the oncogenic potential of activated TEAD signaling .

What emerging roles of TEAD3 can be investigated using these antibodies?

Beyond the canonical Hippo pathway, TEAD3 antibodies have revealed novel functions:

• DNA repair mechanisms:

  • Immunoprecipitation with TEAD3 antibodies identified association with DNA repair proteins including XRCC5, XRCC6, PARP1, and RIF1 .

  • Co-localization studies using TEAD3 antibodies revealed that TEADs localize with DNA damage-induced nuclear foci marked by γH2AX and Rap1-interacting factor 1 .

  • TEAD3 depletion makes cells more susceptible to DNA damage by various agents, promoting genomic instability .

• Cross-pathway interactions:

  • TEAD3 antibodies can be used to investigate how TEAD3 regulates Wnt signaling, as TEADs induce expression of Wnt inhibitors like DKK1 and WNT5A/B .

  • Studies suggest TEADs form a negative feedback loop with Wnt signaling, where TEADs are activated via upstream Wnt pathway while simultaneously inhibiting Wnt signaling .

What are common troubleshooting approaches for TEAD3 antibodies in Western blotting?

When optimizing Western blot detection of TEAD3:

• Sample preparation considerations:

  • Lysates should be freshly made and used immediately to minimize protein degradation .

  • For lower expressing tissues (pancreas, liver), higher protein loading (60 μg) may be required .

• Band detection issues:

  • Expected molecular weight: 49-50 kDa .

  • An additional band at approximately 30 kDa may represent a TEAD3 splice variant .

  • TEAD3 expression varies significantly between tissue types; placenta and JEG-3 cells show strong expression, while HeLa, HepG2, and MCF-7 have lower levels .

• Signal enhancement strategies:

  • Extended exposure times (103-180 seconds) may be needed for tissues with lower expression .

  • Signal amplification using ECL techniques improves detection sensitivity .

  • For higher sensitivity, consider advanced detection systems like ECL Basic Kit with extended exposure times .

How can researchers optimize TEAD3 immunoprecipitation protocols?

For successful TEAD3 immunoprecipitation:

• Starting material recommendations:

  • Use 2 mg of whole cell lysate (e.g., HEK293) for optimal results .

  • TEAD3 antibodies at 1:100-1:200 dilution are typically effective for IP .

• Technical approach:

  • The GeLC-MS approach maximizes detection of low-abundance interactors .

  • For TEAD3 interaction studies, three biological replicates with different batches of cells are recommended .

  • Control and bait IPs should be collected simultaneously to minimize differences in background proteins .

• Validation methods:

  • Western blot the IP product with the same or different TEAD3 antibody at 1:1000 dilution .

  • Include appropriate controls: IgG isotype control IP, input control, and TEAD3 IP sample .

  • Mass spectrometry analysis of IP samples can identify TEAD3-associated proteins and confirm antibody specificity .