WNT10A Antibody, Biotin conjugated

What is WNT10A and why is it important in research?

WNT10A (Wingless-Type MMTV Integration Site Family, Member 10A) is a ligand for members of the frizzled family of seven transmembrane receptors. It functions primarily in the canonical Wnt/β-catenin signaling pathway and is crucial for normal ectoderm development, tooth development, postnatal maintenance of tongue papillae and sweat ducts, and hair follicle function . Research interest in WNT10A has increased due to its involvement in developmental processes and its emerging role in disease states, particularly in oncogenic pathways like renal cell carcinoma .

What are the key applications for WNT10A antibody, biotin conjugated?

The biotin-conjugated WNT10A antibody is primarily used in Western Blotting (WB), Immunohistochemistry (IHC), and ELISA techniques . This antibody has been specifically selected for its ability to recognize WNT10A in immunohistochemical staining and Western blotting applications . The biotin conjugation offers enhanced sensitivity and versatility, allowing researchers to use streptavidin-based detection systems with various reporter molecules. The polyclonal nature of available antibodies (like ABIN1872346) provides recognition of multiple epitopes, enhancing detection sensitivity .

How does the biotin conjugation enhance WNT10A antibody functionality?

Biotin conjugation creates a powerful advantage through the high-affinity interaction between biotin and streptavidin (Kd ≈ 10^-15 M). This allows:

• Enhanced sensitivity through signal amplification

• Greater flexibility in detection methods (fluorescent, chemiluminescent, colorimetric)

• Improved stability of antibody-antigen complexes

• Compatibility with multi-labeling experiments

• Reduced background in certain applications

The WNT10A antibody conjugated to biotin (e.g., ABIN1872346) enables researchers to implement streptavidin-based detection systems without requiring secondary antibodies, streamlining experimental procedures .

What controls should be included when using WNT10A antibody in experiments?

When designing experiments with WNT10A antibody, researchers should include:

• Positive controls: Cell lines or tissues known to express WNT10A (e.g., RAW264.7 cells used in Western blotting or skeletal muscle tissue for IHC)

• Negative controls:

  • Primary antibody omission control

  • Isotype control (matching IgG at the same concentration)

  • Competitive peptide blocking (as demonstrated with sc-69135P peptide for antibody validation)

• Functional controls: When possible, include samples with:

  • WNT10A overexpression (e.g., pcDNA-WNT10A transfection)

  • WNT10A knockdown (e.g., WNT10A siRNA treatment)

These controls validate antibody specificity and help troubleshoot potential technical issues in detection protocols.

What are optimal sample preparation methods for WNT10A antibody, biotin conjugated?

Based on published methodologies, the following sample preparation approaches have been successful:

Optimal antibody dilutions should be determined empirically for each experimental system .

How should researchers optimize WNT10A antibody concentration for different applications?

Optimization requires a systematic titration approach:

• For Western blotting:

  • Begin with a range of antibody concentrations (0.5-5 μg/ml)

  • Testing has shown that 1-2 μg/ml is effective for RAW264.7 cell lysates

  • Monitor signal-to-noise ratio and adjust accordingly

• For IHC/ICC:

  • Start with manufacturer's recommended range (typically 5-10 μg/ml for paraffin sections)

  • Different tissues may require different concentrations (e.g., 5 μg/ml for mouse skeletal muscle vs. 10 μg/ml for human skeletal muscle)

  • Use serial dilutions to determine optimal concentration

• For ELISA:

  • Perform checkerboard titration with both coating and detection antibodies

  • Include standard curves to assess sensitivity and dynamic range

Document optimization parameters for reproducibility in future experiments.

How can WNT10A antibody be used to investigate WNT/β-catenin signaling pathway?

WNT10A antibody can serve as a powerful tool for studying the WNT/β-catenin pathway through:

• Pathway component analysis: Co-detection of WNT10A with pathway components including:

  • β-catenin (particularly nuclear translocation)

  • Downstream targets like cyclin D1 and c-myc

• Integrated experimental approaches:

  • Combine forced WNT10A expression (pcDNA-WNT10A transfection) with β-catenin siRNA knockdown

  • Use WNT10A antibody to confirm expression levels

  • Measure pathway activation using TCF/LEF reporter assays

• Functional consequence assessment:

  • Cell proliferation assays

  • Migration and invasion assays (wound-healing, transwell, Matrigel-coated transwell)

  • Colony formation assays to evaluate transformation capacity

Research has demonstrated that WNT10A activates β-catenin signaling in renal cell carcinoma, with WNT10A overexpression increasing nuclear β-catenin, cyclin D1, and c-myc levels .

What methodological approaches enable investigation of WNT10A in oncogenic processes?

Multiple complementary approaches can be employed:

• Expression profiling:

  • Tissue microarray analysis with WNT10A antibody (1:200 dilution, 2h incubation)

  • Quantification using histoscore (positive cell percentage × intensity)

  • Separate assessment of membranous, cytoplasmic, and nuclear staining

• Functional analysis:

  • Gain-of-function: Transfection with expression vectors (pcDNA-WNT10A)

  • Loss-of-function: siRNA-mediated knockdown

  • Effect on downstream targets using Western blot and immunocytochemistry

• Phenotypic characterization:

  • Cell migration (wound-healing, transwell assays)

  • Cell invasion (Matrigel-coated transwell)

  • Transformation capacity (soft agar colony formation)

• Mechanistic validation:

  • Co-transfection with β-catenin siRNA to determine pathway dependence

  • TCF/LEF reporter assays to quantify canonical pathway activation

These approaches have successfully demonstrated WNT10A's oncogenic role in renal cell carcinoma through β-catenin-dependent signaling .

How can researchers troubleshoot non-specific binding when using WNT10A antibody?

When encountering non-specific binding, researchers should implement these troubleshooting strategies:

• Validation of specificity:

  • Competitive peptide blocking (as performed with sc-69135P)

  • Comparing results from multiple antibodies targeting different WNT10A epitopes

  • Functional validation through overexpression and knockdown experiments

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration (e.g., from 5% to 10% serum)

  • Add detergents (0.1-0.3% Triton X-100) to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Titrate antibody concentration systematically

  • Test different incubation conditions (time, temperature)

• Sample preparation modifications:

  • Optimize fixation protocol

  • Adjust antigen retrieval conditions (buffer pH, duration)

  • Increase washing stringency (duration, detergent concentration)

What are best practices for quantifying WNT10A expression using antibody-based methods?

Robust quantification requires methodological rigor:

• For IHC/ICC quantification:

  • Use established scoring systems such as histoscore (positive cell percentage × intensity)

  • Employ intensity classification: negative, weak, moderate, and strong

  • Separately record membranous, cytoplasmic, and nuclear staining

  • Have multiple blinded observers score samples independently

• For Western blot quantification:

  • Use appropriate loading controls

  • Apply densitometry software with background subtraction

  • Normalize WNT10A bands to loading controls

  • Present data from multiple independent experiments

• For functional readouts:

  • Combine protein detection with functional assays (e.g., TCF/LEF reporter)

  • Correlate WNT10A levels with downstream effects

• Statistical validation:

  • Perform appropriate statistical tests

  • Include sufficient biological and technical replicates

  • Validate findings across multiple experimental approaches

How can researchers interpret contradictory results from WNT10A antibody experiments?

When faced with contradictory data, consider these analytical approaches:

• Antibody-related factors:

  • Compare results using antibodies targeting different WNT10A epitopes

  • Validate specificity through competitive peptide blocking

  • Assess potential post-translational modifications affecting epitope recognition

• Experimental conditions:

  • Evaluate differences in sample preparation (fixation, antigen retrieval)

  • Consider cell/tissue type-specific effects on protein conformation or interacting partners

  • Assess expression levels and detection limits of different methods

• Biological context:

  • WNT10A may have context-dependent functions

  • Consider crosstalk with other signaling pathways

  • Evaluate potential isoform-specific effects

• Integrated data analysis:

  • Triangulate findings using multiple methods (IHC, WB, functional assays)

  • Correlate with genetic data (mutations, expression)

  • Seek validation in independent experimental systems

How do results from WNT10A detection methods compare between different tissue types?

Research demonstrates important considerations for cross-tissue comparisons:

• Antibody concentration requirements vary by tissue:

  • 5 μg/ml optimal for mouse skeletal muscle

  • 10 μg/ml required for human skeletal muscle

  • Different fixation protocols may be necessary for different tissues

• Expression pattern differences:

  • Subcellular localization may vary by tissue type

  • WNT10A expression levels differ between tissues

  • Background staining characteristics can be tissue-specific

• Methodological adaptations:

  • Tissue-specific antigen retrieval optimization

  • Adjusted blocking protocols (5000 μg/ml BSA for some tissues)

  • Varied antibody incubation times (1-2 hours depending on tissue type)

• Validation approaches:

  • Include known positive tissue controls

  • Consider tissue microarrays for standardized comparison

  • Use multiple detection methods when comparing across tissues

How can WNT10A antibody be used to study the impact of WNT10A mutations?

WNT10A mutations have been implicated in odonto-onycho-dermal dysplasia syndrome (OODD) and nonsyndromic tooth agenesis . Research approaches include:

• Mutation characterization:

  • Identify specific mutations using whole-exome sequencing and Sanger sequencing

  • Study biallelic variants such as:

    • c.1176C > A (p.Cys392*)

    • c.812G > A (p.Cys271Tyr)

    • c.637G > A (p.Gly213Ser)

    • c.985C > T (p.Arg329*)

• Antibody-based detection strategies:

  • Select antibodies targeting epitopes preserved in specific mutations

  • For truncation mutations (e.g., p.Cys392*), use N-terminal targeting antibodies

  • For missense mutations (e.g., p.Cys271Tyr), assess effects on epitope recognition

• Functional impact assessment:

  • Compare wild-type vs. mutant protein expression

  • Evaluate effects on protein-protein interactions

  • Assess pathway activation differences (β-catenin signaling)

• Tissue-specific effects:

  • Examine expression in developmental contexts

  • Compare with phenotypic manifestations in patient samples

How can WNT10A antibody be optimized for multi-labeling experiments?

For co-detection of WNT10A with other proteins:

• Biotin-streptavidin system advantages:

  • Compatibility with various detection platforms

  • Signal amplification capability

  • Flexibility in fluorophore selection

• Sequential immunostaining protocol:

  • First primary antibody: non-biotinylated antibody with direct fluorophore detection

  • Second primary antibody: biotinylated WNT10A antibody

  • Detect with streptavidin-conjugated fluorophore with non-overlapping spectrum

• Controls for multi-labeling:

  • Single-stained controls to assess bleed-through

  • Secondary-only controls to evaluate non-specific binding

  • Absorption controls with competing peptides

• Cross-reactivity prevention:

  • Use antibodies from different host species

  • Block between sequential staining steps

  • Consider tyramide signal amplification for particularly low-abundance targets

What novel approaches can integrate WNT10A antibody detection with modern omics techniques?

Advanced integrative approaches include:

• Spatial transcriptomics correlation:

  • Combine WNT10A immunostaining with spatial transcriptomics

  • Correlate protein localization with mRNA expression patterns

  • Identify co-expression networks in specific tissue regions

• Proximity ligation assays (PLA):

  • Use biotinylated WNT10A antibody with antibodies against interaction partners

  • Visualize and quantify protein-protein interactions in situ

  • Map interaction networks spatially within tissues

• ChIP-sequencing applications:

  • Utilize WNT10A antibody for chromatin immunoprecipitation

  • Identify WNT10A-associated DNA regions

  • Correlate with β-catenin binding sites and target gene expression

• Mass spectrometry integration:

  • Immunoprecipitate WNT10A and associated complexes

  • Identify novel binding partners through mass spectrometry

  • Characterize post-translational modifications affecting function