wnt4a Antibody

What is WNT4 and why are specific antibodies needed for its detection?

WNT4 (Wingless-type MMTV integration site family member 4) is a secreted glycoprotein that functions in the Wnt signaling pathway. In humans, the canonical protein consists of 351 amino acid residues with a mass of 39.1 kDa and is primarily localized in the extracellular matrix . WNT4 undergoes post-translational modifications, particularly glycosylation, which can affect epitope recognition . Specific antibodies are required because WNT4 shares structural similarities with other Wnt family members, necessitating reagents that can discriminate between these related proteins. Additionally, up to two different isoforms have been reported for WNT4, requiring antibodies that can either recognize conserved regions or distinguish between variants .

How should researchers select between monoclonal and polyclonal WNT4 antibodies?

The selection depends on your experimental goals:

Monoclonal antibodies (e.g., MAB4751, clone 55025; MAB475, clone 55010; sc-376279, clone B-6):

• Provide consistent lot-to-lot reproducibility for longitudinal studies

• Recognize a single epitope, reducing background but potentially limiting sensitivity

• Often preferred for applications requiring high specificity such as immunohistochemistry

• Example: The MAB4751 monoclonal antibody shows no cross-reactivity with recombinant mouse Wnt-1, 2b, 5a, 5b, 8a, 8b, 9b, 10a, 10b, 11, 16, or recombinant human Wnt-2, 3a, 7a, or 7b in direct ELISAs

Polyclonal antibodies (e.g., CAB7809, 14371-1-AP):

• Recognize multiple epitopes, potentially increasing detection sensitivity

• May detect protein despite partial denaturation or modification

• Useful for applications like Western blotting where protein conformation may be altered

• Example: The 14371-1-AP polyclonal antibody has demonstrated robust detection across multiple applications including WB, IHC, and IF

What species reactivity should be considered when selecting a WNT4 antibody?

WNT4 gene orthologs exist in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee and chicken . When selecting an antibody, consider:

Note that while manufacturers may claim cross-reactivity, validation in your specific model organism is essential. For example, if working with zebrafish (where the gene is sometimes designated as wnt4a), additional validation would be necessary as most commercial antibodies are developed against mammalian epitopes .

What are the optimal protocols for detecting WNT4 using Western blotting?

For successful WNT4 detection by Western blot:

• Sample preparation:

  • Use reducing conditions (presence of DTT or β-mercaptoethanol)

  • Published data indicates successful detection in human breast cancer tissues and cell lines (MCF-7, SK-BR-3, HeLa)

• Gel and transfer parameters:

  • Standard SDS-PAGE (10-12% gels) is suitable

  • PVDF membranes are commonly used for WNT4 detection

• Antibody dilution and detection:

  • Primary antibody: typically 1:500-1:2000 dilution (e.g., 2 μg/mL for MAB4751)

  • Secondary antibody: HRP-conjugated anti-species IgG

  • Expected band size: approximately 39 kDa, though variations between 39-45 kDa have been reported

• Buffer systems:

  • Success has been reported using Immunoblot Buffer Group 1 for MAB4751

What techniques maximize sensitivity for WNT4 immunohistochemistry/immunofluorescence?

For optimal IHC/IF detection of WNT4:

• Tissue preparation:

  • For paraffin sections: immersion fixation in 4% PFA is recommended

  • For IF of cells: fixation in cold 4% PFA has been successful

• Antigen retrieval:

  • For 14371-1-AP: TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative

• Antibody dilutions and incubation conditions:

  • MAB4751: 1-25 μg/mL (typically 1.7-5 μg/mL) for IHC; 3 μg/mL for ICC (3 hours at room temperature)

  • 14371-1-AP: 1:50-1:500 dilution for both IHC and IF/ICC

• Detection systems:

  • For IHC: Anti-Rat HRP-DAB Cell & Tissue Staining Kit or Anti-Rat IgG VisUCyte™ HRP Polymer

  • For IF: Secondary antibodies such as NorthernLights™ 557-conjugated Anti-Rat IgG or appropriate Alexa Fluor conjugates

• Counterstaining:

  • DAPI for nuclear staining in IF

  • Hematoxylin for IHC

How can researchers determine the appropriate WNT4 antibody concentration for their specific application?

Determining optimal antibody concentration requires systematic titration:

• Preliminary range-finding:

  • Start with manufacturer's recommended range (e.g., 1:50-1:500 for IHC with 14371-1-AP)

  • For Western blot: test 3-4 dilutions spanning 1:500-1:2000

  • For IHC/IF: test concentrations between 1-25 μg/mL

• Evaluation criteria:

  • Signal-to-noise ratio (specific staining versus background)

  • Presence of expected subcellular localization (cytoplasmic/membrane for WNT4)

  • Consistency with published observations (e.g., cytoplasmic staining in breast cancer cells)

• Validation experiments:

  • Include positive control tissues with known WNT4 expression (e.g., breast cancer tissue for MAB4751)

  • Consider negative controls (tissues with low/no WNT4 expression or primary antibody omission)

  • For advanced validation, include knockdown/knockout samples where available

How can researchers utilize WNT4 antibodies to investigate developmental processes?

WNT4 plays crucial roles in multiple developmental processes that can be studied using specific antibody-based approaches:

• Heart development:

  • WNT4 is required for ostia development in the Drosophila heart

  • Technique: Combine WNT4 immunostaining with temporal gene expression analysis to track developmental progression

  • Example approach: In Drosophila studies, WNT4 mRNA expression was observed to increase dramatically in ostia progenitors just before ostia formation, with expression levels progressively increasing from anterior to posterior

• β-cell maturation:

  • WNT4 is heterogeneously activated in maturing β-cells and controls insulin secretion capacity

  • Technique: Dual immunofluorescence for WNT4 and β-cell markers can reveal maturation states

  • Research finding: WNT4-positive β-cells are more mature, while WNT4-negative cells retain proliferative capacity

• Kidney development:

  • WNT4 is crucial for kidney development and mesenchymal-epithelial transition

  • Technique: Developmental time-course analysis with WNT4 antibodies can track critical transitions

What technical approaches can resolve heterogeneous WNT4 expression in different cell populations?

Recent research has revealed important heterogeneity in WNT4 expression that requires sophisticated technical approaches:

• Single-cell resolution techniques:

  • Combine WNT4 immunofluorescence with markers for specific cell populations

  • Example: Researchers detected WNT4 heterogeneity in β-cells, revealing significant differences in gene expression profiles between WNT4-high and WNT4-low populations

• Flow cytometry-based separation:

  • Cell sorting based on WNT4 expression levels can isolate distinct subpopulations

  • Findings from β-cell research showed WNT4-high cells had enhanced expression of genes related to β-cell maturation and function, while WNT4-low cells showed enrichment for proliferation markers

• Lineage tracing approaches:

  • Combine Cre-driven lineage tracing with WNT4 antibody staining

  • Research findings showed that while all β-cells eventually express WNT4, the timing varies, creating functional heterogeneity during development

What methodological considerations are important for studying WNT4 post-translational modifications?

WNT4 undergoes important post-translational modifications, particularly glycosylation, which affect its function and detection :

• Epitope selection considerations:

  • Choose antibodies raised against epitopes unlikely to be masked by glycosylation

  • MAB4751 and MAB475 target specific amino acid regions (Ile37-Glu76 and aa 37-76/222-295 respectively) that may be differently affected by modifications

• Deglycosylation experiments:

  • Treatment with glycosidases prior to Western blotting can reveal the impact of glycosylation on antibody detection

  • Compare molecular weight shifts pre- and post-treatment to assess glycosylation extent

• Combining detection methods:

  • Use antibodies recognizing different epitopes to comprehensively detect all WNT4 forms

  • Consider using glycosylation-specific staining in parallel with WNT4 antibodies to assess co-localization

How should researchers address variable molecular weights observed for WNT4 in Western blots?

Researchers frequently observe variable molecular weights for WNT4, typically ranging from 39-45 kDa . This variability may result from:

• Post-translational modifications:

  • Glycosylation is a known modification of WNT4

  • Solution: Include deglycosylated controls to establish the core protein size

• Isoform variation:

  • Up to two different isoforms have been reported for WNT4

  • Approach: Use isoform-specific antibodies or primers (for validation at the RNA level)

• Species differences:

  • Minor variations in molecular weight can occur between species

  • Solution: Always include appropriate species-matched positive controls

• Technical variations:

  • Buffer systems and gel percentage can affect protein migration

  • Recommendation: Standardize protocols and include molecular weight markers

What are essential controls for validating WNT4 antibody specificity?

To ensure the reliability of WNT4 antibody results, implement these validation controls:

• Positive controls:

  • Tissues/cells with known WNT4 expression:

    • Breast cancer tissue and cell lines (MCF-7, SK-BR-3)

    • Kidney tissue (human and mouse)

    • β-cells for developmental studies

• Negative controls:

  • Primary antibody omission

  • Isotype control antibodies (e.g., IgG1 kappa for sc-376279)

  • Tissues with minimal WNT4 expression

• Specificity validation:

  • Peptide competition assays

  • siRNA/CRISPR knockdown of WNT4 (demonstrated effectiveness in β-cell studies)

  • Cross-reactivity testing: MAB4751 shows no cross-reactivity with multiple related Wnt proteins in direct ELISAs

• Orthogonal validation:

  • Confirm protein detection with RNA expression data

  • Use multiple antibodies targeting different epitopes

  • Compare results across different detection methods (WB, IHC, IF)

What approaches can overcome challenges in detecting low levels of WNT4 expression?

WNT4 can be difficult to detect in certain contexts due to low expression levels, as observed in several developmental studies :

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry

  • Enhanced chemiluminescence (ECL) systems with extended exposure for Western blotting

  • Consider using HRP-polymer detection systems as used successfully with MAB4751

• Sample enrichment techniques:

  • Immunoprecipitation prior to Western blotting

  • Cell sorting to enrich for WNT4-expressing populations

  • Subcellular fractionation to concentrate secreted or membrane-associated WNT4

• Alternative detection approaches:

  • In situ hybridization for mRNA detection when protein levels are below antibody detection limits (successful in developmental studies)

  • Proximity ligation assays (PLA) to detect low abundance proteins through interaction partners

  • Consider reporter systems (e.g., GFP) for live cell studies of WNT4 expression

• Timing considerations:

  • WNT4 expression varies temporally during development

  • Example: In Drosophila heart development, WNT4 expression in ostia increased dramatically just before formation, with highest expression in posterior segments

  • In β-cell development, WNT4 expression reaches all cells by P5 (postnatal day 5) despite initial heterogeneity

How can researchers design experiments to investigate WNT4 signaling pathway interactions?

WNT4 functions in both canonical and non-canonical Wnt signaling pathways, requiring specific experimental designs:

• Pathway-specific reporter assays:

  • Canonical Wnt signaling: TOP/FOP flash assays to measure β-catenin-mediated transcription

  • Research observation: Canonical Wnt signaling pathway reporter construct was specifically expressed in ostia cells, and Wnt4 was necessary for high levels of reporter activity

• Co-immunoprecipitation approaches:

  • Use WNT4 antibodies for pull-down experiments to identify interacting proteins

  • Secondary validation with reverse co-IP using antibodies against putative partners

• Pharmacological manipulation:

  • Combine WNT4 immunodetection with pathway inhibitors:

    • Canonical pathway: GSK3β inhibitors (LiCl, CHIR99021)

    • Non-canonical pathway: JNK inhibitors

  • Research finding: Disruption of canonical Wnt signaling pathway, but not non-canonical, phenocopied Wnt4 mutation in Drosophila heart development

• Genetic approaches:

  • Conditional knockout models (e.g., Pdx1-CreER mice for β-cell studies)

  • Validation of phenotypes using WNT4 antibody staining to confirm protein loss

  • Evidence: WNT4 conditional knockout in β-cells led to glucose intolerance as early as 2 weeks after inactivation