WASF1 Antibody, HRP conjugated

What is WASF1/WAVE1 and what is its function in cells?

WASF1/WAVE1 functions as a downstream effector molecule that transmits signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton, promoting actin filament formation . As part of the WAVE complex, it regulates lamellipodia formation through interaction with the Arp2/3 complex . WASF1 is also required for BDNF-NTRK2 endocytic trafficking and signaling from early endosomes, and plays a role in regulating mitochondrial dynamics .

The protein contains a highly conserved C-terminal actin-binding WCA region that includes the WASP-homology 2 (WH2) domain . This region is critical for binding actin and the Arp2/3 complex to promote actin polymerization . Recent studies have identified de novo truncating mutations in WASF1 as causes of intellectual disability with autistic features and seizures, highlighting its importance in neuronal development .

What applications are suitable for WASF1 antibodies, particularly HRP-conjugated versions?

WASF1 antibodies, including HRP-conjugated variants, are suitable for multiple research applications:

• Western Blotting (WB): All WASF1 antibodies in the search results are validated for Western blot . HRP-conjugated antibodies provide direct detection without requiring secondary antibodies, simplifying the protocol and potentially reducing background .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Several antibodies are validated for immunofluorescence applications . For HRP-conjugated antibodies, tyramide signal amplification can be used to generate fluorescent signals.

• Immunohistochemistry (IHC): Many antibodies can be used for IHC, particularly IHC-P (paraffin-embedded) . HRP conjugation is advantageous for chromogenic detection.

• Flow Cytometry: Some antibodies are suitable for intracellular flow cytometry .

• ELISA and Cell-Based ELISA: HRP-conjugated antibodies are particularly useful for ELISA applications , providing direct enzymatic detection.

The optimal application depends on the specific antibody clone, format, and experimental question.

What species reactivity can be expected from WASF1 antibodies?

Based on the provided information, most WASF1 antibodies demonstrate reactivity to:

The cross-species reactivity reflects the high conservation of WASF1 protein sequences across mammalian species, particularly in functional domains like the WCA region . When selecting an antibody for your research, verify the specific reactivity of your chosen antibody, as this can vary between different clones and manufacturers.

What controls should be used when working with WASF1 antibodies?

When designing experiments using WASF1 antibodies, include these essential controls:

• Positive Controls:

  • Brain tissue from mouse or rat

  • Neuronal cell lines like C6

  • K562 cells for flow cytometry

  • Primary cortical neurons for immunofluorescence

• Negative Controls:

  • Isotype control antibody matching the WASF1 antibody's isotype

  • Primary antibody omission control

  • Blocking peptide competition (using the immunogen peptide)

• Loading/Normalization Controls:

  • Beta-tubulin, as demonstrated in immunofluorescence studies

  • GAPDH or actin for Western blot normalization

• Expression Validation:

  • RNA expression data for the same cell lines to validate protein expression patterns

• For Mutation Studies:

  • Include both wild-type and mutant samples, as different antibody epitopes may detect truncated proteins with varying efficiency

These controls ensure reliable interpretation of results and help troubleshoot inconsistent findings.

What is the basic protocol for Western blot using HRP-conjugated WASF1 antibodies?

Based on the search results and standard laboratory practices, here is a protocol for Western blot using HRP-conjugated WASF1 antibodies:

• Sample Preparation:

  • Prepare whole cell extracts (30-40 μg of protein per lane)

  • Successful sample types include brain tissue, C6 cells, HeLa cells, and primary neurons

• SDS-PAGE:

  • Separate proteins using 7.5% SDS-PAGE for optimal resolution of WASF1 (~75 kDa)

• Transfer:

  • Transfer proteins to a PVDF or nitrocellulose membrane

• Blocking:

  • Block the membrane with 5% non-fat milk or BSA in TBST

• Antibody Incubation:

  • For direct detection with HRP-conjugated WASF1 antibody:

    • Dilute according to manufacturer's recommendation (typically 1:1000-1:2000)

    • Incubate for 1-2 hours at room temperature or overnight at 4°C

  • For two-step detection:

    • Incubate with unconjugated WASF1 antibody (1:1000 dilution)

    • Wash and incubate with HRP-conjugated secondary antibody

• Detection:

  • Apply ECL substrate and detect signal using film or digital imaging

  • WASF1 should appear at approximately 75 kDa

  • In samples with mutations, both full-length (75 kDa) and truncated (~70 kDa) proteins may be detected

• Analysis:

  • Perform densitometry for quantification, normalizing to loading controls

  • For mutation studies, compare the ratio of full-length to truncated protein

How should WASF1 antibodies be stored for optimal performance?

For optimal stability and performance of WASF1 antibodies:

• Long-term Storage:

  • Store at -20°C for up to one year

  • Aliquot antibodies to avoid repeated freeze-thaw cycles

• Short-term Storage:

  • Store at 4°C for up to one month if used frequently

• Additional Considerations for HRP-conjugated Antibodies:

  • Protect from light to preserve HRP activity

  • Include preservatives to prevent microbial contamination

  • Avoid oxidizing agents that could affect HRP enzyme function

  • Monitor for precipitation, which may indicate denaturation

• Working Solution Preparation:

  • Dilute in appropriate buffer immediately before use

  • Add protein (BSA) to stabilize diluted antibody

  • Return concentrated stock to recommended storage conditions promptly

Following these guidelines will help maintain antibody performance and extend shelf-life.

How can I optimize my immunofluorescence protocol using WASF1 antibodies?

Based on successful immunofluorescence approaches described in the search results:

• Sample Preparation:

  • For neuronal cells: Use DIV9 rat E18 primary cortical neurons

  • Fixation: 4% paraformaldehyde at room temperature for 15 minutes

  • Permeabilization: 0.1-0.2% Triton X-100 in PBS

• Antibody Concentration:

  • Optimal dilution for WASF1 antibody: 1:500 for neuronal samples

  • For HRP-conjugated antibodies: Use tyramide signal amplification for fluorescent detection

• Co-staining Strategy:

  • WASF1 can be effectively co-stained with cytoskeletal markers like beta-Tubulin 3/Tuj1 (1:500 dilution)

  • Use nuclear counterstain like DAPI

• Signal Enhancement:

  • For low-expressing samples: Consider signal amplification methods

  • Use confocal microscopy for improved signal-to-noise ratio and spatial resolution

• Controls and Validation:

  • Include no-primary-antibody controls

  • Use known positive control tissues (brain sections)

  • Consider peptide competition controls

• Analysis Approaches:

  • Quantify signal intensity relative to background

  • Assess colocalization with interaction partners

  • Evaluate subcellular distribution patterns

This optimization approach should yield reliable, reproducible immunofluorescence results for WASF1 detection.

What is the significance of HRP conjugation in WASF1 antibodies?

HRP (Horseradish Peroxidase) conjugation to WASF1 antibodies offers several methodological advantages:

• Direct Detection System:

  • Eliminates the need for secondary antibodies, simplifying protocols and reducing experiment time

  • Decreases potential background from secondary antibody cross-reactivity

• Signal Amplification:

  • HRP enzymatic activity provides signal amplification, enhancing sensitivity for detecting low-abundance proteins

  • Particularly valuable for detecting endogenous WASF1 in samples with limited expression

• Versatile Detection Options:

  • Compatible with multiple substrates:

    • Colorimetric (DAB, TMB) for IHC and ELISA

    • Chemiluminescent (ECL) for Western blot

    • Fluorescent (TSA) for fluorescence applications

• Quantitative Applications:

  • Enables precise quantification in applications like ELISA and Western blot

  • The Cell-Based ELISA kit can detect WASF1 expression in as few as 5,000 HeLa cells

• Stability Considerations:

  • HRP conjugates typically maintain activity for extended periods when properly stored

  • Enables consistent results across experimental replicates

The search results demonstrate successful use of HRP-conjugated antibodies in WASF1 research, particularly for Western blot detection systems .

How do I troubleshoot inconsistent results when using WASF1 antibodies in neuronal samples?

When encountering variable results with WASF1 antibodies in neuronal preparations, consider these advanced troubleshooting approaches:

• Expression Heterogeneity:

  • Real-time PCR analysis has shown variable WASF1 mRNA levels between different samples, including between individuals with identical mutations

  • Protein levels may not directly correlate with mRNA expression, requiring parallel analysis of both

• Epitope-Specific Detection Strategies:

  • Use multiple antibodies targeting different WASF1 epitopes:

    • N-terminal antibodies (e.g., Sigma-Aldrich W0267)

    • C-terminal antibodies (e.g., Abcam ab50356)

  • This approach is critical for detecting potential truncated variants

• Technical Optimization:

  • For Western blot: Use 7.5% SDS-PAGE for optimal separation

  • For immunofluorescence: 4% paraformaldehyde fixation for 15 minutes at room temperature

  • For Cell-Based ELISA: Seed at least 20,000 adherent cells per well

• Neuronal Stage-Specific Considerations:

  • WASF1 expression and localization may vary with neuronal maturation

  • DIV9 rat E18 primary cortical neurons have been successfully used

• Signal Quantification:

  • Use densitometry for Western blot quantification

  • In patients with WASF1 mutations, truncated protein was detected at only 14-25% of control levels, while full-length protein was at ~50%

• Sample Handling:

  • Include phosphatase inhibitors if studying phosphorylated forms

  • Avoid repeated freeze-thaw cycles of samples

• Cross-Reactivity Assessment:

  • Validate antibody specificity against other WAVE family members

  • Confirm results with genetic knockdown/knockout controls when possible

This systematic approach should help identify and address sources of inconsistency in WASF1 detection.

What are the best experimental conditions to detect post-translational modifications of WASF1?

Detection of WASF1 post-translational modifications requires specialized approaches:

• Phosphorylation-Specific Antibodies:

  • Several antibodies targeting specific phosphorylation sites are available:

    • Anti-WASF1 (pTyr125) antibody

    • Anti-WASF1 (Tyr410) antibody

  • These can be used in Western blot, ELISA, IHC, and IF applications

• Sample Preparation Protocol:

  • Include phosphatase inhibitor cocktails in lysis buffers

  • Consider stimulation conditions that activate regulatory pathways:

    • RAC1 activation (GTPase that regulates WASF1 activity)

    • Growth factor treatment (e.g., BDNF, which requires WASF1 for endocytic trafficking)

• Separation Techniques:

  • Standard approach: Western blot with phospho-specific antibodies

  • Advanced approach: Phos-tag SDS-PAGE for enhanced separation of phosphorylated forms

  • High-resolution approach: 2D gel electrophoresis (IEF followed by SDS-PAGE)

• Detection Strategy:

  • Parallel detection with total and modification-specific antibodies

  • Sequential probing of the same membrane after stripping

  • Multiplex fluorescent detection of total and modified forms

• Validation Methods:

  • Phosphatase treatment as negative control

  • Kinase inhibitor treatment to confirm specificity

  • Mutagenesis of modification sites in expression constructs

• Mass Spectrometry Approaches:

  • Immunoprecipitation followed by MS/MS analysis

  • Enrichment strategies for phosphopeptides

  • Quantitative MS approaches to measure modification stoichiometry

While the search results primarily focus on tyrosine phosphorylation , similar principles can be applied to investigate other modifications including serine/threonine phosphorylation, ubiquitination, and SUMOylation.

How can I distinguish between different WAVE family members using antibodies?

Differentiating between WAVE family proteins requires strategic antibody selection and experimental design:

• Epitope Selection Strategy:

  • Target non-conserved regions between WAVE family members

  • Antibodies against specific amino acid regions (e.g., AA 91-140 ) can provide specificity

  • N-terminal regions generally show lower conservation than the WCA domain

• Molecular Weight Differentiation:

  • Western blot analysis can distinguish family members by size:

    • WASF1/WAVE1: ~75 kDa

    • Other family members may have different molecular weights

  • Use high-resolution gels (7.5% SDS-PAGE) for optimal separation

• Expression Pattern Analysis:

  • WASF1/WAVE1 shows strong expression in brain tissue

  • Compare with known tissue-specific expression patterns of other family members

  • Use tissues with differential expression as positive/negative controls

• Validation Approaches:

  Validation Method	Implementation
  Genetic validation	Use samples from knockout models or siRNA-treated cells
  Immunodepletion	Sequential immunoprecipitation to deplete specific family members
  Recombinant proteins	Test antibody reactivity against purified family members
  Mass spectrometry	Confirm identity of detected bands by peptide analysis

• Functional Correlation:

  • WASF1/WAVE1 has specific roles in neuronal development and function

  • Correlate antibody detection with known functional readouts

  • Assess phenotypic effects of family member-specific manipulations

By combining these approaches, researchers can confidently distinguish WASF1/WAVE1 from other family members in their experimental systems.

What are the considerations for using WASF1 antibodies in patient samples with WASF1 mutations?

Working with patient samples harboring WASF1 mutations presents unique challenges that require specific approaches:

• Epitope Accessibility Strategy:

  • Use antibodies targeting different regions of WASF1:

    • N-terminal antibodies detect both normal and truncated proteins

    • C-terminal antibodies may fail to detect truncated proteins if the epitope is lost

  • This dual-antibody approach provides comprehensive analysis of mutation effects

• Protein Detection Patterns:

  • In patient samples with truncating mutations:

    • Full-length WASF1 (75 kDa) from the wild-type allele

    • Truncated protein (~70 kDa) from the mutant allele

  • Quantitative analysis showed full-length protein at ~50% of control levels and truncated protein at 14-25%

• Mutation-Specific Considerations:

  • Known pathogenic mutations include:

    • c.1516C>T (p.Arg506Ter)

    • c.1558C>T (p.Gln520Ter)

    • c.1482delinsGCCAGG (p.Ile494MetfsTer23)

  • All three variants disrupt the C-terminal actin-binding WCA domain

  • Mutations cluster significantly around the WASP-homology 2 (WH2) domain (p = 1.31 × 10⁻⁶)

• Functional Correlation:

  • Fibroblast cells from affected individuals showed defects in actin remodeling

  • Correlate protein expression patterns with cytoskeletal phenotypes

  • Consider the relationship between protein levels and clinical severity

• Control Selection:

  • Age-matched controls without WASF1 mutations

  • Family members (particularly unaffected siblings)

  • Cell type-matched controls when possible

• Technical Optimizations:

  • Extended run times for SDS-PAGE to resolve closely migrating bands

  • Use of gradient gels for improved separation

  • Advanced imaging systems for detecting subtle differences

These considerations enable accurate characterization of WASF1 mutations and their effects on protein expression and function.

How can I quantitatively analyze WASF1 expression in relation to actin dynamics?

For quantitative analysis of the relationship between WASF1 and actin cytoskeleton:

• Protein Expression Quantification:

  • Western blot with densitometry analysis

  • Cell-Based ELISA for high-throughput screening

  • Flow cytometry for single-cell analysis

• Microscopy-Based Co-localization Analysis:

  • Immunofluorescence co-staining of WASF1 with:

    • Actin (phalloidin staining)

    • Arp2/3 complex components

    • Other cytoskeletal markers like beta-Tubulin 3

  • Quantitative parameters:

    • Pearson's correlation coefficient

    • Manders' overlap coefficient

    • Intensity correlation analysis

• Functional Correlation Methods:

  • Actin polymerization assays

  • Lamellipodia dynamics quantification

  • Cell migration analysis

  • Correlate with WASF1 expression/modification levels

• Experimental Manipulation Approaches:

  Manipulation	Readout	Analysis
  WASF1 knockdown/overexpression	Actin organization	Quantitative image analysis
  Expression of mutant WASF1	Cytoskeletal defects	Morphological assessment
  Treatment with actin-modulating drugs	WASF1 localization	Time-course analysis
  RAC1 activation/inhibition	WASF1-actin interaction	Co-immunoprecipitation

• Advanced Biophysical Approaches:

  • Fluorescence Recovery After Photobleaching (FRAP) to measure dynamics

  • Förster Resonance Energy Transfer (FRET) for protein-protein interactions

  • Super-resolution microscopy for nanoscale organization

• Disease Model Analysis:

  • In samples with WASF1 mutations, truncated protein levels correlated with actin remodeling defects

  • Quantify the relationship between mutant protein expression and cytoskeletal phenotypes

These approaches provide complementary data on how WASF1 expression levels and modifications affect actin cytoskeletal dynamics in both normal and disease states.

How do I design experiments to characterize novel post-translational modifications of WASF1?

To characterize novel post-translational modifications (PTMs) of WASF1, implement a systematic discovery and validation workflow:

• Discovery Phase Techniques:

  • Mass Spectrometry Approaches:

    • Immunoprecipitate WASF1 from cells/tissues of interest

    • Perform tryptic digest and analyze by LC-MS/MS

    • Use enrichment strategies for specific modification types (phospho-enrichment, etc.)

  • Protein Microarrays:

    • Screen with modification-specific detection reagents

    • Identify novel modification sites

• Validation Strategy:

  • Generate modification-specific antibodies

    • Use synthetic modified peptides as immunogens

    • Validate specificity against modified and unmodified WASF1

  • Site-directed mutagenesis:

    • Mutate putative modification sites

    • Assess functional consequences

• Functional Characterization:

  • Signaling Pathway Analysis:

    • Identify stimuli that induce the modification

    • Determine upstream enzymes (kinases, etc.)

    • Map downstream functional effects

  • Structural Impact Assessment:

    • Evaluate effects on WAVE complex formation

    • Analyze impact on interaction with Arp2/3 complex

    • Assess influence on actin polymerization

• Physiological Relevance:

  • Developmental Regulation:

    • Examine modification patterns during neuronal development

    • Correlate with WASF1-dependent processes

  • Disease Association:

    • Compare modification patterns in normal vs. pathological conditions

    • Assess relationship to WASF1-related disorders

• Experimental Controls:

  • Enzymatic Removal:

    • Treat samples with phosphatases, deubiquitinases, etc.

    • Confirm specificity of modification detection

  • Inhibitor Studies:

    • Use specific inhibitors of modifying enzymes

    • Evaluate effects on WASF1 function and localization

• Advanced Techniques:

  • Proximity Labeling:

    • Identify proteins in close proximity to modified WASF1

    • Map modification-dependent interaction networks

  • Single-Molecule Analysis:

    • Assess how modifications affect WASF1 conformational dynamics

    • Measure kinetic parameters of interactions

This comprehensive approach will enable characterization of novel WASF1 modifications and their functional significance in cytoskeletal regulation.

What experimental designs can reveal the role of WASF1 in neurological disorders?

To investigate WASF1's role in neurological disorders, consider these experimental approaches:

• Patient-Derived Models:

  • Fibroblast Analysis:

    • Patients with WASF1 mutations showed defects in actin remodeling

    • Compare cytoskeletal organization between patient and control cells

  • iPSC-Derived Neurons:

    • Reprogram patient cells to induced pluripotent stem cells

    • Differentiate into neurons to study neuron-specific phenotypes

    • Analyze morphology, synaptic function, and electrophysiology

• Genetic Approaches:

  • CRISPR/Cas9 Models:

    • Introduce known pathogenic mutations (e.g., c.1516C>T, p.Arg506Ter)

    • Create isogenic cell lines differing only in WASF1 status

  • Animal Models:

    • Generate mice with patient-specific WASF1 mutations

    • Assess behavioral, cognitive, and neuroanatomical phenotypes

• Molecular Analysis:

  • Protein Expression Patterns:

    • Western blot analysis revealed both full-length and truncated WASF1 in patient samples

    • Quantify expression relative to controls (truncated protein present at 14-25% of control levels)

  • Cellular Distribution:

    • Immunofluorescence analysis in neuronal cells

    • Assess subcellular localization changes caused by mutations

• Functional Assessments:

  • Cytoskeletal Dynamics:

    • Live imaging of actin dynamics in patient-derived neurons

    • Quantify lamellipodia formation and dynamics

  • Synaptic Function:

    • Electrophysiological recordings

    • Synaptic protein localization

    • Dendritic spine morphology analysis

• Molecular Pathway Investigation:

  • WASF1 Complex Analysis:

    • Assess formation of the WAVE Regulatory Complex

    • Determine if mutations affect interaction with RAC1

  • Arp2/3 Interaction:

    • Evaluate binding to Arp2/3 complex

    • Measure actin nucleation activity

• Therapeutic Exploration:

  • Rescue Experiments:

    • Reintroduce wild-type WASF1 in patient cells

    • Test compounds targeting downstream pathways

  • Drug Screening:

    • Screen for compounds that correct cytoskeletal defects

    • Validate in more complex neuronal models

These approaches can establish causal relationships between WASF1 mutations and neurodevelopmental phenotypes, potentially identifying therapeutic targets.

How can I optimize multiplexed detection of WASF1 and its interaction partners?

For effective multiplexed detection of WASF1 and its binding partners:

• Antibody Selection Strategy:

  • Combine antibodies from different host species:

    • Rabbit anti-WASF1

    • Mouse anti-Arp2/3 components

    • Goat anti-actin or cytoskeletal proteins

  • Choose non-overlapping epitopes when using multiple WASF1 antibodies

  • For HRP-conjugated antibodies, use spectrally distinct substrates or sequential detection

• Advanced Imaging Techniques:

  • Multiplexed Immunofluorescence:

    • Use spectrally distinct fluorophores

    • Employ linear unmixing for overlapping spectra

    • Consider multi-round staining with antibody stripping

  • Super-resolution Methods:

    • STORM/PALM for nanoscale co-localization

    • Structured Illumination Microscopy (SIM) for improved resolution

• Protein-Protein Interaction Analysis:

  • Proximity Ligation Assay (PLA):

    • Detect WASF1 interactions with binding partners

    • Generates punctate signal only when proteins are <40nm apart

  • Co-immunoprecipitation:

    • Sequential co-IP to isolate specific complexes

    • Multiplex detection on blots using different visualization methods

• Experimental Design Considerations:

  Approach	Advantage	Challenge	Solution
  Sequential immunoblotting	Uses standard equipment	Incomplete stripping	Validate stripping efficiency
  Multiplex fluorescence	Simultaneous detection	Spectral overlap	Use spectral unmixing
  Multi-epitope detection	Confirms identity	Epitope masking	Try different antibody combinations
  Mass spectrometry	Unbiased detection	Sensitivity	Use enrichment strategies

• Optimization for HRP-Conjugated Antibodies:

  • Use TSA (Tyramide Signal Amplification) with different fluorophores

  • Employ sequential detection with HRP inactivation between rounds

  • Consider enzyme-substrate pairs with different properties (HRP/AP)

• Controls and Validation:

  • Single-antibody controls

  • Blocking peptide competition

  • Genetic manipulation (knockdown/knockout)

  • Split-epitope validation

This systematic approach enables reliable multiplexed detection of WASF1 and its interaction partners, providing insights into the composition and dynamics of WASF1-containing complexes.