DACT3 Antibody

What is DACT3 and what is its primary function in cellular signaling?

DACT3 (Dapper Antagonist of Catenin 3, also known as Dapper Homolog 3) is a member of the DACT gene family that functions primarily as a negative regulator of Wnt/β-catenin signaling pathway. DACT3 mediates this inhibition through interaction with Dishevelled (Dvl) family proteins, thereby blocking β-catenin entry into the nucleus . This regulation is critical during embryonic development and tissue homeostasis, making DACT3 an important research target for developmental biology and cancer studies .

What are the molecular characteristics of DACT3 protein that influence antibody development?

DACT3 is a 65 kDa protein (predicted) with several distinct domains that influence antibody development strategies :

• A coiled-coil region (amino acids 63-87)

• An arginine-rich domain (amino acids 351-476)

• A PDZ-binding motif (amino acids 607-610)

How does DACT3 expression differ across tissue types and species?

Research indicates variable DACT3 expression patterns:

• In humans, DACT3 is expressed in cerebral cortex and uterus

• In AML cells, DACT3 protein levels are significantly reduced compared to normal hematopoietic cells

• Western blot analyses reveal specific bands for DACT3 at approximately 45 kDa in human brain tissue and 50 kDa in mouse brain tissue

When designing experiments, researchers should anticipate these variations and select antibodies validated for their species of interest, as mouse and human DACT3 share 82% amino acid identity over residues 486-587 .

What criteria should guide DACT3 antibody selection for specific experimental applications?

Selection criteria should include:

For reproducible results, researchers should validate the antibody in their experimental system regardless of manufacturer claims.

How can I properly validate a DACT3 antibody for my specific experimental system?

A comprehensive validation protocol should include:

• Positive and negative controls:

  • Use cell lines with known DACT3 expression (e.g., 293F as positive control)

  • Include DACT3 knockout/knockdown samples as negative controls

• Multiple detection methods:

  • Compare results from at least two techniques (e.g., Western blot and immunohistochemistry)

  • Confirm band size matches predicted molecular weight (65 kDa)

• Sensitivity assessment:

  • Test a dilution series to determine optimal antibody concentration

  • For Western blot, recommended dilutions typically range from 1:500 to 1:2000

• Cross-reactivity evaluation:

  • Assess potential cross-reactivity with other DACT family members (DACT1, DACT2)

  • This is particularly important as DACT family members share structural similarities

What are the differences between polyclonal and monoclonal DACT3 antibodies in practice?

What is the optimal protocol for detecting DACT3 by Western blot?

Based on validated research protocols :

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • For brain tissue samples, specialized extraction protocols may be required due to high lipid content

• Electrophoresis conditions:

  • Load 15-20 μg of total protein per lane

  • Use reducing conditions for optimal epitope exposure

  • Employ Immunoblot Buffer Group 8 for consistent results

• Transfer and detection:

  • Transfer to PVDF membrane (preferred over nitrocellulose for DACT3)

  • Block with 5% non-fat dry milk in TBST

  • Incubate with primary DACT3 antibody at dilutions of 1:500-1:2000

  • For polyclonal antibodies: use 1 μg/mL concentration

  • Detect with appropriate HRP-conjugated secondary antibody (Anti-Goat IgG for sheep-derived antibodies, Anti-Rabbit IgG for rabbit-derived antibodies)

• Expected results:

  • Human DACT3: approximately 45 kDa band

  • Mouse DACT3: approximately 50 kDa band

  • Potential additional bands may represent splice variants or post-translational modifications

How can I optimize DACT3 immunohistochemistry protocols for fixed tissue samples?

Optimized protocol for DACT3 immunohistochemistry:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde

  • For paraffin-embedded sections, perform antigen retrieval using citrate buffer (pH 6.0)

  • For frozen sections, acetone fixation maintains antigen integrity

• Antibody incubation:

  • Block with 5% normal serum from the same species as the secondary antibody

  • Incubate with DACT3 antibody (1:100-1:500 dilution) overnight at 4°C

  • For brain tissues, extend incubation time to ensure penetration

• Signal development:

  • Use biotinylated secondary antibody followed by streptavidin-HRP for signal amplification

  • Develop with DAB substrate for permanent preparations or fluorescent conjugates for co-localization studies

• Controls and interpretation:

  • Include positive controls (e.g., normal brain tissue)

  • Evaluate cellular localization (expected to be primarily cytoplasmic)

  • Quantify expression using digital image analysis software

What approaches can be used to study DACT3 protein-protein interactions in the Wnt signaling pathway?

Several methodological approaches can elucidate DACT3 interactions:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate with DACT3 antibody and probe for Dishevelled or other potential interactors

  • Use mild lysis conditions to preserve protein complexes

  • Consider cross-linking to stabilize transient interactions

• Proximity ligation assay (PLA):

  • Allows visualization of DACT3-Dvl interactions in situ

  • Requires antibodies from different species for DACT3 and its interaction partner

  • Provides quantitative assessment of interaction frequency

• Fluorescence resonance energy transfer (FRET):

  • Tag DACT3 and potential partners with appropriate fluorophores

  • Enables real-time monitoring of interactions in living cells

  • Requires careful controls to account for spectral overlap

• Bimolecular fluorescence complementation (BiFC):

  • Split fluorescent protein fragments are fused to DACT3 and interacting proteins

  • Interaction brings fragments together to form functional fluorophore

  • Less prone to artifacts than FRET but irreversible once formed

How can I distinguish between epigenetic mechanisms regulating DACT3 expression in different cancer types?

DACT3 regulation involves complex epigenetic mechanisms that differ between cancer types:

• Integrated analysis approach:

  • In colorectal cancer, DACT3 repression involves bivalent histone modifications rather than DNA methylation

  • In AML, downregulation involves both DNA methylation and histone deacetylation

• Experimental protocol:

  • Perform chromatin immunoprecipitation (ChIP) with antibodies against specific histone marks (H3K4me3, H3K27me3)

  • Combine with bisulfite sequencing to assess DNA methylation status

  • Compare effects of different epigenetic drugs:

    • DZNep (histone methylation inhibitor)

    • TSA (histone deacetylase inhibitor)

    • 5-AzaC (DNA methyltransferase inhibitor)

  • In colorectal cancer, DZNep/TSA combination robustly induces DACT3

  • In AML, combined chidamide (HDAC inhibitor) and azacytidine (DNMT inhibitor) upregulates DACT3

• Data interpretation:

  • Changes in DACT3 expression following treatment with specific epigenetic modulators can identify the predominant regulatory mechanism

  • Compare effects across multiple cell lines to establish tissue-specific patterns

Why do Western blots sometimes show multiple bands when probing for DACT3, and how should these be interpreted?

Multiple bands in DACT3 Western blots may result from:

• Biological factors:

  • Splice variants: A potential splice variant with 45 aa substitution for residues 413-610 has been reported

  • Post-translational modifications: Phosphorylation may cause mobility shifts

  • Species differences: Human DACT3 (45 kDa) vs. mouse DACT3 (50 kDa)

  • Frameshift in human DACT3 at position 119 yielding a 41 kDa product

• Technical considerations:

  • Protein degradation during sample preparation

  • Non-specific antibody binding

  • Insufficient blocking or washing

• Validation strategy:

  • Use recombinant DACT3 as positive control

  • Compare patterns across multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

  • Use DACT3 knockout/knockdown samples to identify non-specific bands

How can I optimize DACT3 antibody-based assays for low-abundance samples?

For samples with low DACT3 expression:

• Sample enrichment techniques:

  • Immunoprecipitate DACT3 before Western blotting

  • Use tissue or cell fractionation to concentrate cellular compartments where DACT3 is localized

• Signal amplification methods:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry

  • Use highly sensitive ECL substrates for Western blot

  • Consider biotin-streptavidin systems for enhanced detection

• Optimization strategies:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Adjust blocking conditions to reduce background while preserving signal

  • Test different antibody concentrations in narrow ranges around manufacturer recommendations

  • For Western blots, larger sample volumes (up to 50 μg) may be needed

• Alternative detection methods:

  • Consider mass spectrometry-based approaches for very low abundance

  • Use proximity extension assay (PEA) for ultrasensitive protein detection

How can DACT3 antibodies be used to assess the therapeutic potential of epigenetic modulators in acute myeloid leukemia?

DACT3 expression serves as a valuable biomarker for epigenetic therapy response in AML:

• Experimental design:

  • Baseline assessment of DACT3 expression in patient samples using validated antibodies

  • Treatment of AML cell lines with epigenetic modulators (HDAC inhibitors, DNMT inhibitors)

  • Time-course analysis of DACT3 re-expression using Western blot and qRT-PCR

  • Correlation of DACT3 re-expression with cellular phenotypes (apoptosis, differentiation)

• Clinical correlation protocol:

  • Stratify AML patients based on DACT3 expression levels

  • Compare treatment outcomes between DACT3-low and DACT3-high groups

  • Monitor DACT3 expression changes during treatment as potential response biomarker

• Mechanistic investigations:

  • Assess correlation between DACT3 re-expression and Wnt/β-catenin pathway inhibition

  • Evaluate cell sensitivity to conventional chemotherapeutics (e.g., adriamycin) after DACT3 restoration

  • Target validation through DACT3 overexpression or knockdown experiments

Research shows DACT3 re-expression correlates with cell apoptosis via inhibition of Wnt/β-catenin signaling in AML, suggesting promising therapeutic prospects, particularly in FLT3-mutant AML .

What methodologies can distinguish the functions of different DACT family members in cancer research?

The DACT family includes three members (DACT1, DACT2, DACT3) with potentially overlapping functions:

• Differential expression analysis:

  • Use specific antibodies for each DACT family member

  • In AML, DACT3 shows significantly decreased expression compared to DACT1 and DACT2

  • Western blot analysis with antibodies specific to each family member can reveal distinct expression patterns

• Functional discrimination techniques:

  • Generate specific knockdowns/knockouts for each family member

  • Perform rescue experiments with individual DACT proteins

  • Conduct domain-swapping experiments to identify functional regions

• Epigenetic regulation differences:

  • DACT2 expression is induced by treatments containing DNA methylation inhibitor 5-AzaC

  • DACT3 expression is strongly induced by combined DZNep/TSA treatment

  • DACT1 shows only modest response to epigenetic modulators

  • These differences can be leveraged to selectively modulate individual family members

• Pathway analysis:

  • Assess effects on different branches of Wnt signaling (canonical vs. non-canonical)

  • Analyze differential protein interaction networks using co-IP followed by mass spectrometry

How can DACT3 antibodies contribute to understanding the relationship between epigenetic regulation and cancer progression?

DACT3 represents an important model for studying epigenetic regulation in cancer:

• Chromatin state mapping:

  • Perform ChIP-seq for histone modifications at DACT3 promoter across cancer progression stages

  • Correlate with DACT3 protein expression using validated antibodies

  • In colorectal cancer, DACT3 promoter shows bivalent histone modifications (activating H3K4me3 and repressive H3K27me3)

• Functional consequences assessment:

  • Restore DACT3 expression through epigenetic modulators

  • Measure effects on Wnt/β-catenin target genes using reporter assays

  • Assess phenotypic changes (proliferation, apoptosis, migration)

  • In AML, DACT3 restoration inhibits leukemia cell growth and improves adriamycin sensitivity

• Clinical correlation studies:

  • Analyze DACT3 expression patterns in patient samples at different disease stages

  • In AML, DACT3 expression increases during complete remission and decreases again at relapse

  • Low DACT3 expression correlates with higher white blood cell counts, higher bone marrow blasts, and FLT3/DNMT3A mutations

• Therapeutic response prediction:

  • Develop immunohistochemistry-based scoring systems for DACT3 expression

  • Correlate scores with response to conventional and epigenetic therapies

  • Use as companion diagnostic for epigenetic-targeting drugs

This methodological framework enables researchers to use DACT3 antibodies as tools for understanding broader principles of epigenetic regulation in cancer biology and developing potential therapeutic approaches.