DVL3 Antibody, HRP conjugated

What is DVL3 and why is it important in research?

DVL3 (Dishevelled segment polarity protein 3) is a critical component of the Wnt signaling pathway, which plays essential roles in cellular processes including proliferation, differentiation, and embryonic development. It functions primarily in the cytoplasm and is involved in transducing signals from frizzled receptors to downstream effectors, influencing cellular responses during development and tissue homeostasis. Research on DVL3 is particularly important because dysregulation of the Wnt pathway has been implicated in numerous diseases, including cancer and neurodegenerative disorders .

What is the difference between unconjugated DVL3 antibody and HRP-conjugated versions?

The primary difference lies in detection methodology. Unconjugated DVL3 antibodies (such as the mouse monoclonal 4D3 clone) require a secondary antibody step for detection in assays like Western blotting. In contrast, HRP-conjugated DVL3 antibodies have horseradish peroxidase directly attached to the antibody molecule, eliminating the need for secondary antibody incubation. This direct conjugation reduces protocol time, decreases background signal by eliminating cross-reactivity from secondary antibodies, and often provides enhanced sensitivity for detection of low-abundance proteins . The choice between conjugated and unconjugated versions should be based on your specific experimental design, detection system availability, and sensitivity requirements.

How can I verify the specificity of DVL3 antibody in my experimental system?

Verifying antibody specificity is crucial for reliable results. For DVL3 antibody, consider these validation approaches:

• Positive and negative controls: Use tissues or cell lines known to express high levels of DVL3 (positive control) and those with low or no expression (negative control).

• Knockdown/knockout validation: Compare antibody signal in wild-type cells versus DVL3 knockdown or knockout cells. Several repositories offer knockout-validated DVL3 antibodies .

• Phosphorylation state analysis: For studying specific phosphorylated forms of DVL3, use phospho-specific antibodies alongside phosphatase treatments as controls .

• Western blot molecular weight verification: Confirm that the detected band appears at the expected molecular weight (approximately 78 kDa for human DVL3) .

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide (amino acids 607-704 for the 4D3 clone) to demonstrate binding specificity .

What is the optimal protocol for Western blotting using HRP-conjugated DVL3 antibody?

For optimal Western blotting results with HRP-conjugated DVL3 antibody, follow this protocol:

• Sample preparation: Lyse cells in cold lysis buffer supplemented with protease inhibitors, phosphatase inhibitors, DTT (0.1 mM), and NaF (10 mM) to preserve phosphorylation states crucial for DVL3 activity assessment .

• Gel electrophoresis: Use 8-10% SDS-PAGE gels for optimal resolution of DVL3 (78 kDa).

• Transfer: Transfer proteins to PVDF membrane (recommended over nitrocellulose for phospho-proteins).

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute HRP-conjugated DVL3 antibody (1:1000 to 1:2000) in blocking buffer and incubate overnight at 4°C.

• Washing: Wash 3-4 times with TBST, 5-10 minutes each.

• Detection: Apply ECL substrate directly (no secondary antibody needed) and image using a chemiluminescence imaging system.

For detecting mobility shifts due to phosphorylation events, consider using 6% gels to achieve better separation of phosphorylated species, as DVL3 phosphorylation status significantly affects its electrophoretic mobility .

How can I optimize the detection of DVL3 phosphorylation states using the HRP-conjugated antibody?

Detecting DVL3 phosphorylation requires specific considerations:

• Phosphatase inhibitors: Always use fresh phosphatase inhibitors in lysis buffers to preserve phosphorylation status.

• Control experiments: Include CK1ε-overexpressing samples as positive controls, as CK1ε is a known kinase for DVL3 .

• Inhibitor treatments: Consider using CK1δ/ε inhibitors (like PF-670642) as negative controls to demonstrate specificity of phosphorylation events .

• Gel percentage: Use lower percentage gels (6-8%) to resolve the mobility shifts caused by phosphorylation.

• Phos-tag™ gels: For enhanced separation of phosphorylated species, consider using Phos-tag™ acrylamide gels.

• Complementary approaches: Pair Western blotting with phospho-specific antibodies (such as anti-Ser643-phosphorylated DVL3) for confirmation of specific phosphorylation sites .

• Quantification: For accurate quantification, normalize phospho-signal to total DVL3 protein levels using ImageJ or similar software .

How should I store and handle HRP-conjugated DVL3 antibody to maintain optimal activity?

Proper storage and handling of HRP-conjugated DVL3 antibody is essential for maintaining its activity:

• Short-term storage: For immediate use within two weeks, store at 4°C .

• Long-term storage: Divide the antibody solution into small aliquots (minimum 20 μL) and freeze at -20°C or preferably -80°C to avoid freeze-thaw cycles .

• Working dilutions: Prepare fresh working dilutions on the day of the experiment.

• Avoid repeated freeze-thaw cycles: Each cycle can reduce antibody activity by approximately 20%.

• Protect from light: HRP is light-sensitive; minimize exposure to bright light during handling.

• Preservatives: Note that many commercial preparations contain antimicrobial agents like ProClin; consider this if you experience cell toxicity in functional assays .

• Reconstitution: If lyophilized, reconstitute using sterile buffer according to manufacturer's instructions and allow full rehydration before use.

How can I use DVL3 antibody to investigate Wnt signaling pathway activation status?

DVL3 phosphorylation serves as a valuable indicator of Wnt pathway activation. To investigate this:

• Electrophoretic mobility shift: Upon Wnt activation, DVL3 becomes hyperphosphorylated, resulting in slower migration on SDS-PAGE. HRP-conjugated DVL3 antibody can directly detect this mobility shift .

• Subcellular localization: Combine Western blotting with subcellular fractionation to track DVL3 relocalization upon Wnt stimulation. This requires separate cytoplasmic and membrane fractions.

• Phospho-specific detection: Use phospho-specific antibodies in conjunction with general DVL3 antibody to monitor specific phosphorylation sites. For example, Ser643 phosphorylation is associated with CK1ε activity on DVL3 .

• Temporal analysis: Design time-course experiments (0-24 hours) after Wnt stimulation to track the dynamic changes in DVL3 phosphorylation state.

• Downstream targets: Monitor β-catenin stabilization and TCF/LEF reporter activity alongside DVL3 phosphorylation to correlate DVL3 modifications with pathway output .

• Inhibitor studies: Use selective inhibitors of upstream (CK1ε inhibitors) or downstream components to dissect the pathway mechanisms in relation to DVL3 function .

What approaches can resolving contradictory results when using DVL3 antibody in different experimental systems?

When facing contradictory results across experimental systems, consider these approaches:

• Isoform specificity: Verify which DVL isoform (DVL1, DVL2, or DVL3) predominates in your experimental system, as they may have differential expression and function. Use RT-qPCR to quantify relative expression of each isoform.

• Phosphorylation dynamics: Confirm whether contradictory results might stem from differential phosphorylation states. Mass spectrometry analysis has identified >50 phosphorylated Ser and Thr residues on DVL3, many of which are constitutively phosphorylated while others are dynamically regulated .

• Cell type specificity: Different cell types may express different levels of kinases (like CK1ε) that modify DVL3. Include positive controls from established DVL3-expressing systems.

• Antibody epitope accessibility: The epitope recognized by the 4D3 clone (amino acids 607-704) may be differentially accessible depending on protein conformation or interaction partners. Try denaturing versus native conditions.

• Cross-validation: Use multiple DVL3 antibodies recognizing different epitopes to confirm results. The PCRP-DVL3-1B10 antibody recognizes amino acids 397-504, providing an alternative detection region .

• Functional readouts: Complement protein detection with functional assays such as TCF/LEF reporter assays or secondary axis formation in Xenopus embryos to correlate protein detection with biological activity .

How can I differentiate between effects of Frizzled receptor activation and CK1ε kinase activity on DVL3 using the HRP-conjugated antibody?

Distinguishing between Frizzled-induced and CK1ε-induced effects on DVL3 is important as they operate through different mechanisms:

• Mutational analysis: Previous research has shown that mutations in the PDZ domain (S280A and S311A) reduce TCF/LEF transcriptional activation but do not affect Frizzled-induced mobility shift or subcellular localization. Conversely, mutations in C-terminal Ser/Thr clusters prevent CK1ε-induced mobility shift but not Frizzled5-induced changes .

• Temporal analysis: CK1ε and Frizzled5 induce different temporal patterns of DVL3 phosphorylation. Design time-course experiments (12-24 hours) to track these different patterns.

• Inhibitor studies: Use CK1δ/ε inhibitors like PF-670642 to specifically inhibit CK1ε-mediated phosphorylation while leaving Frizzled-induced effects intact .

• Co-immunoprecipitation: Use agarose-conjugated DVL3 antibody to pull down DVL3 complexes and analyze binding partners under Frizzled activation versus CK1ε overexpression conditions.

• Phospho-specific antibodies: Use phospho-specific antibodies (such as anti-Ser643) that recognize CK1ε-specific phosphorylation sites .

• Mass spectrometry: For comprehensive analysis, perform MS/MS identification of phosphorylation sites under Frizzled activation versus CK1ε overexpression .

What methodologies can be combined with DVL3 antibody detection to comprehensively analyze Wnt pathway components?

For comprehensive Wnt pathway analysis, combine DVL3 antibody detection with:

• Dual luciferase reporter assays: Measure TCF/LEF transcriptional activity using reporters like TOPFlash/FOPFlash to correlate DVL3 modifications with functional outcomes .

• Immunoprecipitation: Use agarose-conjugated DVL3 antibody to pull down interaction partners and analyze complex formation under different conditions .

• Mass spectrometry: Identify all phosphorylation sites and their dynamics using MS/MS analysis of immunoprecipitated DVL3 under various stimulation conditions .

• Immunocytofluorescence: Track subcellular localization changes using fluorescent-conjugated DVL3 antibodies or unconjugated primary followed by fluorescent secondary antibodies .

• CRISPR/Cas9 gene editing: Create DVL3 knockout or knock-in cell lines to study loss-of-function or specific mutations.

• Proximity ligation assay (PLA): Visualize in situ protein-protein interactions between DVL3 and other Wnt pathway components.

• ChIP-seq: When combined with TCF/LEF antibodies, can identify genomic targets downstream of DVL3-mediated Wnt signaling.

• Phosphatase treatments: Apply lambda phosphatase to samples to confirm that mobility shifts are due to phosphorylation rather than other post-translational modifications.

What are common issues when using HRP-conjugated DVL3 antibody in Western blotting and how can they be resolved?

When using HRP-conjugated DVL3 antibody, researchers may encounter several issues:

• Multiple bands or smears: This could reflect various phosphorylation states of DVL3. Confirm by treating samples with phosphatase. If the problem persists, optimize sample preparation by using fresh protease inhibitors and keeping samples cold throughout processing.

• Weak or no signal: Increase antibody concentration, extend incubation time, or use enhanced chemiluminescence substrate. Verify protein transfer efficiency using Ponceau S staining.

• High background: Increase washing duration and frequency, optimize blocking conditions, or dilute the antibody further. For HRP-conjugated antibodies, ensure the substrate is freshly prepared.

• Inconsistent results between experiments: Standardize lysate preparation, protein quantification, and gel loading. Use internal loading controls and maintain consistent experimental conditions.

• Non-specific bands: Perform antibody validation using knockout or knockdown samples. If necessary, pre-adsorb the antibody with the immunizing peptide.

• Unexpected molecular weight: DVL3 runs at approximately 78 kDa, but phosphorylation can cause significant upward mobility shifts. If bands appear at unexpected sizes, verify with positive controls and consider the possibility of splice variants or proteolytic fragments.

How should I interpret changes in DVL3 electrophoretic mobility in relation to its phosphorylation status?

Interpreting DVL3 electrophoretic mobility changes requires understanding the relationship between phosphorylation and migration patterns:

• PS-DVL (phosphorylated and shifted DVL): The upper, slower-migrating band represents hyperphosphorylated DVL3, often associated with active Wnt signaling or CK1ε overexpression .

• Migration patterns: DVL3 with mutations in the C-terminal Ser/Thr clusters prevents CK1ε-induced mobility shift, suggesting these regions are critical for the PS-DVL formation .

• Quantification approach: When quantifying phosphorylation using the mobility shift, calculate the ratio of shifted (PS-DVL) to total DVL3 signal using densitometry software like ImageJ .

• Kinase specificity: Different kinases may induce distinct mobility patterns. CK1ε typically causes more pronounced shifts than other kinases .

• Time-dependent changes: Following Wnt stimulation, the appearance of PS-DVL typically precedes downstream events like β-catenin stabilization.

• Differential effects: Frizzled5 and CK1ε can both induce mobility shifts but through different mechanisms and involving different phosphorylation sites .

• Additional considerations: Very high levels of phosphorylation can sometimes result in diffuse bands rather than discrete shifted bands. In such cases, Phos-tag™ gels may provide better resolution.

What controls should be included when studying DVL3 phosphorylation in Wnt signaling experiments?

When studying DVL3 phosphorylation in Wnt signaling, include these essential controls:

• Positive phosphorylation control: Cells overexpressing CK1ε, which induces robust DVL3 phosphorylation and mobility shift .

• Negative phosphorylation control: Samples treated with CK1 inhibitors like PF-670642 (10 μM) to prevent DVL3 phosphorylation .

• Pathway activation control: Cells treated with Wnt3a or other canonical Wnt ligands to induce physiological pathway activation.

• Pathway inhibition control: Cells treated with Wnt pathway inhibitors (e.g., Dkk1 or IWP compounds) to demonstrate specificity.

• Phosphatase treatment control: Treat duplicate samples with lambda phosphatase to confirm that mobility shifts are phosphorylation-dependent.

• Dominant negative control: Include samples expressing dominant negative CK1ε (P3 mutant) to demonstrate kinase-specific effects .

• Loading control: Include housekeeping proteins (β-actin, GAPDH) for normalization across samples.

• Functional readout control: Parallel analysis of TCF/LEF reporter activity to correlate DVL3 phosphorylation with transcriptional outcomes .

How can DVL3 antibodies be used to investigate the role of DVL3 in cancer and other pathological conditions?

DVL3 antibodies are valuable tools for investigating pathological conditions:

• Expression level analysis: Use HRP-conjugated DVL3 antibody to compare DVL3 expression levels between normal and cancer tissues via Western blotting or immunohistochemistry.

• Phosphorylation status: Analyze DVL3 phosphorylation patterns in different cancer types, as abnormal Wnt pathway activation is a hallmark of many cancers .

• Drug screening: Use DVL3 phosphorylation as a readout for high-throughput screening of compounds that modulate Wnt signaling in cancer models.

• Biomarker development: Investigate the potential of DVL3 expression or specific phosphorylation patterns as prognostic or predictive biomarkers in cancer.

• Therapeutic target validation: Combine antibody-based detection with small molecule inhibitors or siRNA approaches targeting DVL3 to evaluate its potential as a therapeutic target.

• Patient-derived samples: Apply DVL3 antibodies to analyze patient samples to correlate DVL3 status with clinical outcomes and treatment responses.

• Neurodegenerative disorders: Investigate DVL3's role in conditions like Alzheimer's disease, where Wnt signaling dysregulation has been implicated .

What emerging technologies can enhance the utility of DVL3 antibodies in research?

Several emerging technologies can expand DVL3 antibody applications:

• Single-cell Western blotting: Analyze DVL3 expression and phosphorylation at the single-cell level to understand cellular heterogeneity.

• Multiplexed immunofluorescence: Combine DVL3 detection with other Wnt pathway components to visualize pathway status in individual cells within complex tissues.

• Super-resolution microscopy: Apply techniques like STORM or PALM with fluorophore-conjugated DVL3 antibodies to visualize subcellular localization at nanometer resolution.

• Tissue clearing techniques: Use DVL3 antibodies with CLARITY, iDISCO, or other clearing methods to visualize DVL3 expression in intact three-dimensional tissues.

• Antibody-based proteomics: Employ DVL3 antibodies in proximity labeling approaches (BioID, APEX) to map the DVL3 interactome under different conditions.

• Microfluidic antibody-based assays: Develop lab-on-chip applications for real-time monitoring of DVL3 status in response to stimuli.

• Single-molecule pull-down: Combine DVL3 antibodies with single-molecule techniques to analyze stoichiometry and composition of individual DVL3-containing complexes.

• CRISPR-based tagging: Use CRISPR/Cas9 to introduce epitope tags into endogenous DVL3 loci for improved antibody detection without overexpression artifacts.

How can phospho-specific DVL3 antibodies complement standard DVL3 antibodies in research applications?

Phospho-specific DVL3 antibodies provide complementary information to standard antibodies:

• Site-specific analysis: Unlike general mobility shift detection, phospho-specific antibodies (such as anti-Ser643) pinpoint exactly which residues are modified under different conditions .

• Temporal resolution: Track phosphorylation of specific sites with greater temporal resolution to establish the sequence of phosphorylation events during Wnt signaling.

• Functional correlation: Link specific phosphorylation events to distinct functional outcomes. For example, PDZ domain phosphorylation (Ser280, Ser311) affects TCF/LEF activation while C-terminal phosphorylation affects subcellular localization .

• Kinase-specific effects: Distinguish between phosphorylation events mediated by different kinases that may target distinct residues on DVL3.

• Quantitative analysis: Perform quantitative Western blotting with phospho-specific antibodies normalized to total DVL3 for precise phosphorylation stoichiometry determination.

• Pathway crosstalk: Investigate how other signaling pathways may influence DVL3 function through phosphorylation at different sites.

• Therapeutic targeting: Design and evaluate inhibitors that specifically prevent phosphorylation at functionally critical residues.

• Multiplexed detection: Combine multiple phospho-specific antibodies in multiplexed assays to obtain a comprehensive phosphorylation profile of DVL3 under different conditions.