WASF3 Antibody, HRP conjugated

What is WASF3 and why is it significant in research?

WASF3 is a member of the Wiskott-Aldrich syndrome protein family that functions as a key regulator of actin cytoskeleton rearrangement. It plays a critical role in cellular processes such as cell motility and invasion. WASF3 has gained significant research interest because its dysregulation has been implicated in various diseases, particularly cancer, where increased expression correlates with more aggressive tumor behavior . As a mediator of cell motility, invasion, and metastasis, WASF3 represents a potential therapeutic target, making antibodies against this protein valuable tools for understanding disease mechanisms .

What cellular localization patterns should researchers expect when using WASF3 antibodies?

When utilizing WASF3 antibodies in immunolabeling experiments, researchers should expect to observe specific subcellular localizations including: cytoskeleton, extracellular exosomes, glial cell projections, glutamatergic synapses, and postsynaptic structures. Additionally, WASF3 is found in SCAR complexes . Proper validation of antibody specificity is essential, as the distribution pattern may vary depending on cell type and experimental conditions. The expected molecular weight of WASF3 is approximately 55kDa, which should be confirmed in Western blot applications to ensure antibody specificity.

How can researchers validate the specificity of WASF3 antibodies in their experimental systems?

To validate WASF3 antibody specificity, researchers should implement a multi-approach strategy:

• Western blot analysis using positive control samples (e.g., mouse brain or rat brain tissues as indicated in specifications)

• Comparison with WASF3 knockout or knockdown samples as negative controls

• Peptide competition assays using the immunogen peptide

• Cross-validation using different antibody clones or detection methods

• Co-localization studies with other markers of known WASF3-associated structures

For HRP-conjugated antibodies specifically, researchers should also perform enzyme activity controls to ensure the conjugate is functioning as expected.

What are the optimal experimental conditions for using HRP-conjugated WASF3 antibodies in Western blot applications?

For optimal results with HRP-conjugated WASF3 antibodies in Western blot applications, researchers should follow these methodological guidelines:

• Sample preparation: Ensure complete protein denaturation using appropriate buffers containing SDS and reducing agents.

• Protein loading: Load 15-30 μg of total protein per lane.

• Dilution ratio: Start with a 1:500 - 1:2000 dilution as recommended , but optimize for your specific antibody and sample.

• Blocking buffer: Use 5% non-fat dry milk or 3-5% BSA in TBST.

• Incubation time: Overnight at 4°C for primary antibody (or 1-2 hours at room temperature).

• Washing steps: Perform at least 3-5 washes with TBST between antibody incubations.

• Detection system: Use enhanced chemiluminescence (ECL) compatible with HRP for visualization.

• Expected band size: Look for a band at approximately 55kDa .

If signal strength is insufficient, consider signal amplification methods compatible with HRP detection systems or extending substrate incubation time.

How can researchers effectively use WASF3 antibodies for investigating hypoxia-induced changes in WASF3 expression?

To effectively investigate hypoxia-induced changes in WASF3 expression using antibodies, researchers should implement the following methodological approach:

• Experimental design: Set up parallel cultures in normoxic and hypoxic conditions (typically 1% O₂) for 24-48 hours as performed in published studies .

• Controls: Include HIF1A inhibitors (such as YC-1) or HIF1A siRNA knockdown samples to confirm hypoxia-specific effects .

• Sample collection: Harvest cells directly in hypoxic chambers to prevent reoxygenation effects.

• Protein analysis: Perform Western blot using WASF3 antibodies alongside HIF1A antibodies as hypoxia markers.

• Quantification: Use densitometry analysis normalized to appropriate loading controls.

• Phosphorylation assessment: As hypoxia increases not only WASF3 expression but also its phosphorylation , consider using phospho-specific antibodies if available.

Research has shown that exposure to hypoxic conditions increases WASF3 expression levels in breast cancer cell lines including MDA231, SKBR3, and MCF7 , providing useful positive controls for such experiments.

What methodological considerations are important when using WASF3 antibodies in immunohistochemistry applications?

When performing immunohistochemistry (IHC) with WASF3 antibodies, researchers should consider the following methodological approaches:

For HRP-conjugated antibodies specifically, substrate optimization (DAB exposure time) is critical for balancing signal intensity and background.

How can researchers design experiments to investigate the relationship between WASF3 and HIF1A in tumor progression?

To investigate the WASF3-HIF1A relationship in tumor progression, researchers should implement a comprehensive experimental approach:

• Expression correlation analysis:

  • Perform parallel Western blots and qRT-PCR for both WASF3 and HIF1A across tumor cell lines

  • Analyze public databases (TCGA, GEO) for WASF3-HIF1A expression correlation in patient samples

• Regulatory mechanism studies:

  • Conduct ChIP assays using anti-HIF1A antibodies to confirm binding to HRE elements in the WASF3 promoter

  • Perform luciferase reporter assays using WASF3 promoter constructs containing HIF1A binding sites under normoxic and hypoxic conditions

  • Use HIF1A inhibitors (YC-1) or siRNA knockdown to verify HIF1A-dependent regulation

• Functional studies:

  • Compare invasion and migration capabilities using scratch wound assays in cells with WASF3 knockdown versus control cells under hypoxic conditions

  • Assess phosphorylation status of WASF3 in response to hypoxia, as phosphoactivation is required for its pro-invasion function

• In vivo models:

  • Develop xenograft models with HIF1A or WASF3 knockdown to evaluate metastatic potential

  • Use immunohistochemistry with WASF3 antibodies to analyze expression patterns in hypoxic tumor regions (identified by pimonidazole staining)

This multi-faceted approach will provide insights into how hypoxia-induced WASF3 expression contributes to tumor progression mechanisms.

What are the optimal experimental controls for WASF3 antibody validation in studies involving its role in metastasis?

For rigorous validation of WASF3 antibodies in metastasis studies, researchers should incorporate these advanced controls:

• Genetic controls:

  • WASF3 knockout or knockdown cell lines (using CRISPR-Cas9 or siRNA)

  • Cells with reconstituted WASF3 expression (rescue experiments)

  • Cells expressing mutant WASF3 lacking key phosphorylation sites

• Expression gradient controls:

  • Panel of cell lines with varying endogenous WASF3 expression levels

  • Inducible WASF3 expression systems to create controlled expression gradients

• Functional controls:

  • Parallel analysis of known WASF3-regulated processes (actin dynamics, MMP production)

  • Correlation of antibody signal with functional metastasis assays (invasion, migration)

• Specificity controls:

  • Pre-absorption with immunizing peptide

  • Cross-reactivity assessment with other WASF family members (WASF1, WASF2)

  • Immunoprecipitation followed by mass spectrometry validation

• Technical controls:

  • Multiple antibody detection methods (fluorescence, enzymatic)

  • Isotype-matched control antibodies

  • Secondary-only controls for HRP-conjugated antibodies

This comprehensive validation strategy ensures that observed phenotypes are specifically attributed to WASF3 rather than antibody artifacts.

How can researchers interpret contradictory results when studying WASF3 expression across different cancer types?

When faced with contradictory results regarding WASF3 expression across cancer types, researchers should implement this analytical framework:

• Technical considerations:

  • Evaluate antibody specificity using Western blot and immunoprecipitation validation

  • Assess epitope accessibility in different sample types (fresh-frozen vs. FFPE)

  • Compare results from multiple detection methods (IHC, Western blot, qRT-PCR)

• Biological heterogeneity analysis:

  • Consider tumor heterogeneity and microenvironmental factors (hypoxia levels vary across tumors)

  • Evaluate WASF3 in context of molecular subtypes of each cancer

  • Assess correlation with hypoxia markers (HIF1A, CA9) in each cancer type

• Regulatory context evaluation:

  • Analyze HIF1A expression and activity in different tumor types

  • Examine status of WASF3 promoter (methylation, mutations) affecting HIF1A binding

  • Investigate post-translational modifications affecting WASF3 stability or detection

• Data integration approaches:

  • Perform meta-analysis of published studies

  • Correlate findings with public database information

  • Consider single-cell analysis to resolve cellular heterogeneity

• Functional validation:

  • Test invasion/migration phenotypes following WASF3 manipulation across cancer types

  • Assess phosphorylation status which determines WASF3 activity rather than just expression levels

This structured approach helps researchers distinguish true biological differences from technical artifacts when interpreting contradictory WASF3 expression patterns.

What mechanisms regulate WASF3 expression in response to hypoxia in cancer cells?

The regulation of WASF3 expression under hypoxic conditions involves several molecular mechanisms:

• HIF1A-mediated transcriptional activation:

  • The WASF3 promoter contains multiple Hypoxia Response Elements (HREs) that conform to the canonical A/G/CGTG sequence

  • Four putative HREs have been identified in the human WASF3 upstream region: three clustered between -27 and -79 bp (HRE1, HRE2, HRE3) and one more distal between -721 and -725 bp (HRE4)

  • ChIP assays have confirmed direct binding of HIF1A to these HRE elements in the WASF3 promoter

  • Luciferase reporter assays using WASF3 promoter constructs demonstrate increased activity under hypoxic conditions

• HIF isoform specificity:

  • HIF1A knockdown, but not HIF2A knockdown, reduces hypoxia-induced WASF3 expression

  • Treatment with the HIF1A-specific inhibitor YC-1 prevents hypoxia-mediated induction of WASF3

• Post-translational regulation:

  • Hypoxia not only increases WASF3 expression but also enhances its phosphorylation

  • Phosphoactivation of WASF3 is required for its pro-invasion function

• mRNA stability regulation:

  • Unlike some hypoxia-responsive genes, WASF3 mRNA stability does not appear to be significantly altered between normoxic and hypoxic conditions

These findings provide a mechanistic explanation for the observation of elevated WASF3 expression in advanced-stage tumors, which often experience hypoxic environments.

How can researchers effectively use WASF3 antibodies to study its role in cancer cell invasion and metastasis?

To effectively study WASF3's role in cancer cell invasion and metastasis using antibodies, researchers should implement this comprehensive experimental approach:

• Expression analysis across metastatic progression:

  • Compare WASF3 levels between primary tumors and matched metastatic lesions

  • Correlate expression with clinical outcomes and metastatic potential

  • Use immunohistochemistry with validated WASF3 antibodies on tissue microarrays

• Functional assays integrated with antibody-based detection:

  • Real-time monitoring of WASF3 localization during invasion using immunofluorescence

  • Correlation of WASF3 phosphorylation status with invasive capacity

  • Assessment of WASF3 in invadopodia formations using confocal microscopy

• Protein interaction studies:

  • Co-immunoprecipitation with WASF3 antibodies to identify binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Immunofluorescence co-localization with actin cytoskeleton components

• Microenvironmental context evaluation:

  • Analysis of WASF3 expression in hypoxic tumor regions using dual immunostaining

  • Correlation with markers of epithelial-mesenchymal transition

  • Assessment of WASF3 in tumor-stroma interface regions

• Therapeutic targeting assessment:

  • Monitoring WASF3 expression/phosphorylation following experimental therapies

  • Using WASF3 antibodies as readouts in drug screening platforms

  • Developing antibody-drug conjugates targeting WASF3-expressing cells

This integrated approach leverages antibody-based detection within functional contexts to comprehensively characterize WASF3's role in the metastatic cascade.

What are the most reliable methods for quantifying changes in WASF3 phosphorylation status in response to hypoxia?

To reliably quantify hypoxia-induced changes in WASF3 phosphorylation status, researchers should consider these methodological approaches:

For optimal results, researchers should:

• Establish hypoxia chambers with precise O₂ control (1-2%)

• Include time-course analyses (e.g., 2h, 6h, 24h, 48h)

• Compare results across multiple detection methods

• Include phosphorylation-deficient WASF3 mutants as negative controls

• Correlate phosphorylation changes with functional invasion assays

This multi-method approach provides robust quantification of WASF3 phosphorylation changes under hypoxic conditions, which is critical since phosphorylation is required for WASF3's invasion-promoting functions.

What are common technical challenges when using HRP-conjugated WASF3 antibodies and how can researchers address them?

Researchers commonly encounter several technical challenges when working with HRP-conjugated WASF3 antibodies. Here are evidence-based solutions for each issue:

• High background in Western blots:

  • Increase blocking stringency (5% BSA instead of milk for phospho-detection)

  • Optimize antibody dilution (test serial dilutions from 1:500 to 1:5000)

  • Increase washing duration and number of washes (5x 10-minute washes)

  • Use fresh transfer buffers and high-quality membranes

  • Include 0.05-0.1% Tween-20 in antibody diluent

• Weak or absent signal:

  • Verify protein transfer efficiency with reversible staining

  • Increase protein loading (up to 50μg per lane)

  • Reduce stringency of washing steps

  • Use enhanced chemiluminescence with extended exposure times

  • Check sample preparation method (ensure phosphatase inhibitors are included for phospho-detection)

• Multiple bands or unexpected band sizes:

  • Validate with positive control samples (mouse or rat brain)

  • Include WASF3 knockdown controls

  • Optimize gel percentage to better resolve proteins around 55kDa

  • Use freshly prepared samples to prevent degradation

  • Consider the possibility of post-translational modifications or splice variants

• HRP activity loss:

  • Store antibody according to manufacturer recommendations (typically 4°C, do not freeze)

  • Avoid repeated freeze-thaw cycles

  • Use antibody aliquots to minimize exposure

  • Check expiration date and storage conditions

  • Include positive control for HRP activity

• Cross-reactivity with other WASF family members:

  • Use peptide competition assays with the immunizing peptide

  • Perform parallel detection with antibodies specific to other WASF family members

  • Confirm specificity with WASF3-null cells

These approaches help researchers overcome technical limitations and achieve reliable results when using HRP-conjugated WASF3 antibodies.

How can researchers optimize protocols for detecting WASF3 in different types of cancer tissues?

Optimizing WASF3 detection across different cancer tissues requires systematic protocol adjustments:

• Tissue-specific fixation optimization:

  • For soft tissues (breast, lung): 12-24h fixation in 10% NBF

  • For dense tissues (colon, pancreas): 24-48h fixation

  • Consider tissue-specific fixatives for special cases

• Antigen retrieval customization:

  • Test multiple methods (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Optimize retrieval duration (10-30 minutes)

  • Adjust heat source (microwave, pressure cooker, or water bath)

• Antibody optimization matrix:

  • Create a dilution series (1:100, 1:200, 1:500, 1:1000)

  • Test various incubation conditions (1h RT, overnight 4°C)

  • Evaluate different detection systems (polymer vs. avidin-biotin)

• Cancer-specific considerations:

  • For tissues with high endogenous peroxidase (liver): Extended peroxidase quenching

  • For tissues with high background (melanoma): Additional blocking steps

  • For hypoxic tumors: Parallel staining with HIF1A to correlate expression

• Signal amplification strategies:

  • Tyramide signal amplification for low-expressing tissues

  • Polymer-based detection systems for enhanced sensitivity

  • Biotin-free systems for tissues with high endogenous biotin

• Validation across cancer types:

  • Include tissue-specific positive controls

  • Perform parallel Western blots from tissue lysates

  • Consider multiplex approaches (WASF3 with lineage markers)

This systematic approach enables reliable WASF3 detection across diverse cancer tissues while accounting for their unique biological and physical properties.

How should researchers interpret and troubleshoot discrepancies between WASF3 mRNA and protein expression data?

When researchers encounter discrepancies between WASF3 mRNA and protein levels, they should apply this analytical framework:

• Technical verification:

  • Confirm primer specificity for mRNA detection (BLAST search, melt curve analysis)

  • Validate antibody specificity using Western blot and knockdown controls

  • Check reference genes/loading controls for normalization

  • Use multiple detection methods for both protein (Western blot, IHC, ELISA) and mRNA (qRT-PCR, RNA-seq)

• Post-transcriptional regulation assessment:

  • Investigate microRNA regulation of WASF3 (miRNA prediction tools and validation)

  • Measure mRNA stability through actinomycin D chase experiments

  • Assess translational efficiency using polysome profiling

• Post-translational regulation analysis:

  • Measure protein half-life through cycloheximide chase assays

  • Investigate ubiquitination status and proteasome dependency

  • Assess phosphorylation status which might affect antibody recognition

• Biological context consideration:

  • Evaluate hypoxic status of samples (HIF1A levels correlate with WASF3)

  • Consider cell cycle phase (potential fluctuations in expression)

  • Assess stress conditions that might alter translation globally

• Experimental design refinement:

  • Include time-course analyses (mRNA changes may precede protein changes)

  • Analyze subcellular fractions (protein might relocalize rather than change in total level)

  • Consider tissue heterogeneity (micro-dissection for enriching specific cell types)

This comprehensive approach enables researchers to determine whether discrepancies represent true biological regulation or technical artifacts, leading to more accurate interpretation of WASF3 expression data.