WASF3 Antibody, Biotin conjugated

What is WASF3 and why is it an important research target?

WASF3 (also known as WAVE3) is a protein that functions as an important mediator of cell motility, invasion, and metastasis. It is expressed at high levels in some advanced stage tumors and participates in actin cytoskeleton reorganization . Recent research has also revealed WASF3's role in mitochondrial function, where it localizes to mitochondria and regulates respiratory supercomplex assembly . The multifaceted roles of WASF3 in cancer progression and cellular metabolism make it a significant target for research across various disciplines including oncology, cell biology, and mitochondrial research.

What are the key specifications of WASF3 Antibody, Biotin conjugated?

The WASF3 Antibody, Biotin conjugated is a polyclonal antibody derived from rabbit hosts that reacts with human WASF3 protein. Its specifications include:

The biotin conjugation enables high-sensitivity detection in various immunoassays without the need for secondary antibodies .

What experimental controls should be implemented when using WASF3 antibody in research?

For rigorous experimental design with WASF3 antibody, researchers should implement multiple controls:

• Positive control: Include lysates from cells known to express WASF3 (e.g., MDA-MB-231, SKBR3, or MCF7 breast cancer cell lines) .

• Negative control: Use WASF3 knockdown cells, which show no response to hypoxia in WASF3-related experiments .

• Isotype control: Employ a non-specific rabbit IgG at equivalent concentrations to assess non-specific binding.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity of detected signals.

• Cross-reactivity assessment: Test the antibody against related WASF family members (WASF1, WASF2) to ensure specificity when studying WASF3.

These controls help validate experimental findings and ensure the observed effects are specifically related to WASF3 rather than technical artifacts .

How can WASF3 antibody be optimized for detecting mitochondrial localization of WASF3?

Recent research has revealed that WASF3 localizes to mitochondria and regulates respiratory supercomplex assembly . To optimize detection of mitochondrial WASF3:

• Subcellular fractionation: Implement differential centrifugation protocols to isolate pure mitochondrial fractions. Verification of fractionation quality should be performed using established mitochondrial markers (e.g., VDAC, COX IV).

• Immunofluorescence co-localization: Co-stain with mitochondrial markers (MitoTracker dyes or antibodies against mitochondrial proteins) and analyze using confocal microscopy with appropriate resolution settings.

• Proximity ligation assay (PLA): Employ PLA to detect interactions between WASF3 and known mitochondrial proteins like ATAD3A or respiratory complex components.

• Mitochondrial import assays: Use isolated mitochondria to assess direct interaction of WASF3 with the mitochondrial import machinery.

• Blue native polyacrylamide gel electrophoresis (BN-PAGE): As demonstrated in research, BN-PAGE can separate different supercomplexes with defined stoichiometry and detect WASF3 association with respiratory supercomplexes after immunoblotting .

The signal specificity should be validated using WASF3-deficient mitochondria as negative controls.

What are the optimal methods for studying WASF3-CYFIP1 interactions using this antibody?

The interaction between WASF3 and CYFIP1 is critical for WASF3 stability and function . To study this interaction:

• Co-immunoprecipitation: Use the biotin-conjugated WASF3 antibody for pull-down assays with avidin-coated beads, followed by immunoblotting for CYFIP1. Research has shown that disruption of this interaction leads to destabilization of the WASF3 complex .

• Proximity ligation assay: This technique can visualize protein-protein interactions in situ with high specificity and sensitivity.

• FRET/BRET analysis: When combined with fluorescently tagged proteins, these techniques allow real-time monitoring of protein interactions.

• Quantitative analysis of complex stability: As demonstrated in published research, treatments that disrupt the WASF3-CYFIP1 interaction (such as stapled peptides) can be measured by pull-down assays to assess complex disruption in a concentration-dependent manner .

• Functional validation: Knockout or knockdown of CYFIP1 has been shown to suppress WASF3 protein levels without affecting transcript levels, confirming that CYFIP1 is essential for WASF3 protein stability .

How can researchers effectively study WASF3 phosphorylation status using this antibody?

WASF3 phosphorylation is crucial for its activation and function in cell motility and invasion . To study phosphorylation:

• Immunoprecipitation followed by phospho-specific detection:

  • Immunoprecipitate WASF3 using the biotin-conjugated antibody

  • Perform immunoblotting with anti-phosphotyrosine antibodies

  • Quantify the ratio of phospho-WASF3 to total WASF3 to assess activation status

• Phosphatase treatment controls:

  • Include samples treated with phosphatases to confirm specificity of phosphorylation signals

  • Compare untreated vs. phosphatase-treated samples to validate phosphorylation-specific bands

• Stimulation experiments:

  • Expose cells to hypoxia, growth factors, or other stimuli known to activate WASF3

  • Monitor changes in phosphorylation status over time

  • Research has shown that hypoxia enhances both WASF3 expression and phosphorylation

• Kinase inhibitor studies:

  • Use specific kinase inhibitors to identify the signaling pathways involved in WASF3 phosphorylation

  • Correlate changes in phosphorylation with functional outcomes such as cell motility

These approaches provide comprehensive analysis of WASF3 phosphorylation dynamics and their functional significance.

What are common issues when using WASF3 antibody in immunoprecipitation studies and how can they be resolved?

Researchers may encounter several challenges when using WASF3 antibody for immunoprecipitation:

• Low yield of immunoprecipitated protein:

  • Optimize antibody concentration (typical range: 1-5 μg per reaction)

  • Extend incubation time (4-16 hours at 4°C)

  • Consider cross-linking the antibody to beads to prevent antibody contamination in eluted samples

  • For biotin-conjugated antibodies, ensure fresh avidin-coated beads are used

• High background or non-specific binding:

  • Increase washing stringency gradually (adjust salt concentration from 150mM to 300mM NaCl)

  • Add non-ionic detergents (0.1-0.5% NP-40 or Triton X-100)

  • Pre-clear lysates with protein G beads or avidin beads before immunoprecipitation

  • Include carrier proteins (BSA) at 0.1-0.5% in wash buffers

• Disruption of protein-protein interactions:

  • Use milder lysis conditions (avoid ionic detergents)

  • Optimize salt concentration (typically 120-150mM is optimal for maintaining interactions)

  • Include protease and phosphatase inhibitors fresh in all buffers

  • Consider chemical crosslinking before lysis for transient interactions

• Interference from biotin in cell culture media:

  • Grow cells in biotin-free media for 24-48 hours before experiments

  • Wash cells extensively to remove residual biotin before lysis

Research has shown that optimized pull-down assays with biotin-conjugated antibodies can successfully detect WASF3-CYFIP1 interactions in a concentration-dependent manner .

How can researchers optimize western blot protocols for detecting WASF3 with high specificity?

To achieve optimal western blot results with WASF3 antibody:

• Sample preparation optimization:

  • Use appropriate lysis buffers containing protease inhibitors

  • For phosphorylated WASF3 detection, include phosphatase inhibitors

  • Optimize protein loading (typically 20-40 μg of total protein)

• Gel selection and transfer parameters:

  • Use 7.5-10% polyacrylamide gels for optimal resolution of WASF3 (~75 kDa)

  • For detecting supercomplexes, use gradient gels (4-12%)

  • Optimize transfer time and voltage based on protein size (longer transfer times for larger complexes)

• Blocking and antibody incubation:

  • Test different blocking agents (5% milk vs. 3-5% BSA)

  • BSA is often preferred for phospho-specific detection

  • Optimize primary antibody dilution (typically 1:500 to 1:2000)

  • Incubate at 4°C overnight for best signal-to-noise ratio

• Signal development considerations:

  • For biotin-conjugated antibodies, use streptavidin-HRP or streptavidin-fluorophore conjugates

  • Optimize substrate exposure time to avoid signal saturation

  • Consider using fluorescent western blot systems for more quantitative analysis

• Validation methods:

  • Include positive controls (e.g., 3T3 cells and 293 cells have been validated)

  • Use WASF3 knockdown samples as negative controls

Published research has successfully used these approaches to detect endogenous WASF3 in various cell types .

What procedures should be followed to validate the specificity of WASF3 antibody in a new experimental system?

When introducing WASF3 antibody into a new experimental system, comprehensive validation is essential:

• Genetic validation:

  • Compare signal between wild-type and WASF3 knockdown/knockout cells

  • Use siRNA or shRNA targeting different regions of WASF3 mRNA

  • Employ CRISPR-Cas9 to generate knockout cell lines for definitive controls

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (10-100× molar excess)

  • Compare signal between blocked and unblocked antibody

  • Specific signals should be significantly reduced or eliminated

• Cross-reactivity assessment:

  • Test antibody against recombinant WASF family proteins (WASF1, WASF2, WASF3)

  • Analyze signal in cells with differential expression of WASF family members

  • Perform immunoblotting after immunoprecipitation to confirm specificity

• Multi-technique concordance:

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

  • Results should be consistent across techniques for true positives

  • Discrepancies might indicate technical issues or context-dependent epitope accessibility

• Species cross-reactivity verification:

  • Test antibody on samples from different species if cross-reactivity is claimed

  • Compare sequences of the immunogen region across species

  • Validate in each species independently

Research has demonstrated that properly validated WASF3 antibodies detect endogenous levels of total WASF3 protein in both human and mouse cells .

How should researchers interpret changes in WASF3 expression under hypoxic conditions?

Research has established that WASF3 expression is regulated by hypoxia through HIF1A . When interpreting hypoxia-induced changes:

• Expression level analysis:

  • Quantify both mRNA and protein levels to distinguish transcriptional vs. post-transcriptional regulation

  • Normalize to appropriate housekeeping genes/proteins stable under hypoxia

  • Compare results across multiple time points (acute vs. chronic hypoxia)

  • Research has shown increased WASF3 expression in MDA-MB-231, SKBR3, and MCF7 cells under hypoxic conditions

• Phosphorylation status assessment:

  • Analyze both total and phosphorylated WASF3

  • Calculate phospho-WASF3/total WASF3 ratio to determine activation status

  • Studies have demonstrated increased phosphorylation of WASF3 in cells exposed to hypoxia

• Functional correlation:

  • Monitor downstream effects such as MMP expression changes

  • Assess cell motility using scratch wound assays

  • Compare invasion potential between normoxic and hypoxic conditions

  • Evidence indicates increased motility in hypoxic cells correlates with WASF3 activation

• HIF1A-dependency validation:

  • Use HIF1A inhibitors or knockdown to confirm mechanism

  • Perform ChIP assays to verify HIF1A binding to WASF3 promoter HREs

  • Utilize reporter assays with WASF3 promoter constructs to confirm transcriptional regulation

These approaches provide comprehensive understanding of hypoxia-mediated WASF3 regulation in experimental systems.

How can WASF3 antibody be used to investigate its role in mitochondrial respiratory complex assembly?

Recent research has uncovered WASF3's novel role in mitochondrial function . To investigate this role:

• Supercomplex assembly analysis:

  • Isolate intact mitochondria using differential centrifugation

  • Perform blue native polyacrylamide gel electrophoresis (BN-PAGE)

  • Immunoblot with WASF3 antibody to detect association with respiratory complexes

  • Research has identified a specific ~720-kDa band representing WASF3 association with respiratory supercomplex (SC) III2+IV

• In-gel activity assays:

  • Follow BN-PAGE with in-gel assays for complex IV activity

  • Compare activity between wild-type and WASF3-overexpressing or transgenic samples

  • Studies have shown ~50% reductions in SC III2+IV in-gel COX activity in WASF3 Tg mice and WASF3-transfected cells

• Co-localization studies:

  • Perform subcellular fractionation to confirm mitochondrial enrichment of WASF3

  • Use confocal microscopy with mitochondrial markers

  • Research has demonstrated WASF3 enrichment in mitochondrial fractions of C2C12 myoblasts

• Protein-protein interaction analysis:

  • Identify WASF3 interactions with respiratory complex components

  • Investigate connections to known mitochondrial proteins like ATAD3A and GRP78

  • Studies have shown WASF3 associates with contact sites of mitochondria and endoplasmic reticulum

• Functional consequences assessment:

  • Measure oxygen consumption rates in cells with altered WASF3 expression

  • Analyze mitochondrial membrane potential using fluorescent probes

  • Quantify ATP production to connect structural findings to bioenergetic outcomes

These methodologies enable comprehensive analysis of WASF3's mitochondrial functions beyond its established cytoskeletal roles.

What considerations are important when designing experiments to study WASF3-CYFIP1 interactions in cancer progression?

The WASF3-CYFIP1 interaction is critical for cancer cell invasion and metastasis . When designing experiments to study this interaction:

• Cell line selection criteria:

  • Choose highly invasive cancer cell lines (e.g., MDA-MB-231 breast cancer, PC3 prostate cancer)

  • Include cell lines with varying metastatic potential for comparative studies

  • Consider patient-derived xenografts or primary tumor cells for clinical relevance

• Intervention strategies:

  • Use genetic approaches (shRNA, CRISPR) targeting CYFIP1 or WASF3

  • Employ peptide-based interventions (e.g., stapled peptides targeting the α-helical interface)

  • Research has shown that stapled peptides (WAHMs) can disrupt the WASF3-CYFIP1 complex and suppress invasion

• Functional readouts:

  • Motility assays (wound healing, single-cell tracking)

  • 3D invasion assays (Matrigel, collagen)

  • Matrix metalloproteinase expression and activity measurements

  • Studies have demonstrated that CYFIP1 knockdown reduces invasion potential similar to WASF3 knockdown

• Molecular mechanism analysis:

  • Monitor complex formation using co-immunoprecipitation

  • Assess protein stability of WASF3 after CYFIP1 depletion

  • Research has shown that CYFIP1 knockdown leads to destabilization of WASF3 protein without affecting transcript levels

• In vivo validation:

  • Design xenograft studies with cells modified to disrupt WASF3-CYFIP1 interaction

  • Assess primary tumor growth versus metastatic spread

  • Use imaging techniques to track metastasis in real-time

These experimental considerations enable comprehensive investigation of how WASF3-CYFIP1 interactions contribute to cancer progression and potential therapeutic interventions.

How can WASF3 antibody be used to investigate the relationship between WASF3 and matrix metalloproteinases in invasion processes?

Research has established connections between WASF3 and matrix metalloproteinases (MMPs) in cancer invasion . To investigate this relationship:

• Expression correlation analysis:

  • Quantify WASF3 and MMP levels (particularly MMP-1) in various experimental conditions

  • Compare expression patterns in normoxic versus hypoxic conditions

  • Research has shown that hypoxia increases both WASF3 and MMP mRNA levels in MDA-MB-231 cells

• Mechanistic studies:

  • Perform WASF3 knockdown/overexpression and measure resulting changes in MMP expression

  • Use chromatin immunoprecipitation to investigate whether WASF3 directly or indirectly regulates MMP promoters

  • Employ pathway inhibitors to identify signaling intermediates between WASF3 and MMP regulation

• Functional invasion assays:

  • Conduct Matrigel invasion assays with MMP inhibitors in WASF3-expressing cells

  • Use MMP activity assays (zymography) to correlate WASF3 levels with functional MMP activity

  • Research has demonstrated that WASF3 knockdown downregulates specific MMPs, explaining the loss of invasion phenotype

• In vivo validation:

  • Utilize mouse models with modulated WASF3 expression

  • Measure MMP levels and activity in primary tumors and metastatic sites

  • Correlate findings with invasion and metastasis metrics

These approaches provide comprehensive insights into the WASF3-MMP regulatory axis in cancer invasion processes.

What experimental design is recommended for investigating potential therapeutic targeting of WASF3 in cancer models?

Based on WASF3's established role in cancer progression, several experimental designs can assess its therapeutic targeting:

• Target validation studies:

  • Compare invasion and metastasis between WASF3-positive and WASF3-knockout models

  • Conduct rescue experiments to confirm specificity

  • Assess correlation between WASF3 levels and clinical outcomes in patient samples

• Intervention approaches:

  • Stapled peptides targeting WASF3-CYFIP1 interaction

    • Research has demonstrated that WAHM (WASF Helix Mimics) peptides targeting the α-helical interface between WASF3 and CYFIP1 can suppress WASF3 activation and reduce invasion

  • Small molecule screening for compounds disrupting WASF3 complexes

  • RNA-based therapeutics (siRNA, antisense oligonucleotides)

• Efficacy assessment:

  • In vitro: Invasion assays, cell motility measurements, MMP activity

  • Ex vivo: Patient-derived organoids or explants

  • In vivo: Metastasis models (e.g., tail vein injection, orthotopic implantation)

• Combination strategies:

  • Test WASF3 targeting with standard chemotherapeutics

  • Explore synergy with anti-angiogenic therapies (given WASF3's regulation by hypoxia)

  • Investigate combinations with immunotherapies

• Resistance mechanisms:

  • Develop models of acquired resistance to WASF3 targeting

  • Identify compensatory pathways activated upon WASF3 inhibition

  • Design rational combination approaches to overcome resistance

These experimental approaches provide a comprehensive framework for developing and evaluating WASF3-targeted therapeutics in cancer.