DVL3 Antibody, Biotin conjugated

What is DVL3 and what is its functional significance in cellular signaling?

DVL3 (Dishevelled, dsh homolog 3) is a critical scaffold protein that plays an essential role in the Wnt signaling pathway. It functions primarily in the cytoplasm, where it transduces signals from frizzled receptors to downstream effectors. This protein is fundamental to various cellular processes including cell proliferation, differentiation, and embryonic development. DVL3 has a calculated molecular weight of 78 kDa and consists of 716 amino acids . The protein's critical role in development is highlighted by its importance in the formation of supermolecular complexes that are essential for proper Wnt/β-catenin canonical signaling pathway activation . Understanding DVL3 is particularly important because dysregulation of the Wnt pathway has been implicated in numerous diseases, including cancer and neurodegenerative disorders .

What are the primary applications for DVL3 antibodies in research?

DVL3 antibodies have been validated for multiple research applications, providing versatile tools for investigating this protein's expression, localization, and interactions. The primary applications include Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), and Enzyme-Linked Immunosorbent Assay (ELISA) . These applications allow researchers to examine DVL3 expression patterns in different tissues, visualize its subcellular localization, study its interactions with other proteins, and quantify its expression levels. Biotin-conjugated DVL3 antibodies specifically enhance detection sensitivity through avidin-biotin complex formation and are particularly valuable in multi-labeling experiments where traditional antibody combinations might produce cross-reactivity .

What sample types have been validated for use with DVL3 antibodies?

DVL3 antibodies have been extensively tested and validated across multiple sample types. Positive Western blot detection has been confirmed in several cell lines including A549 cells, MCF-7 cells, and Raji cells . For immunohistochemistry applications, these antibodies have successfully detected DVL3 in human colon cancer tissue, human prostate cancer tissue, mouse colon tissue, and human cervical cancer tissue . Immunofluorescence applications have been validated in HeLa cells, while immunoprecipitation has been confirmed in Raji cells . The antibodies demonstrate reactivity with human and mouse samples, with cited reactivity also including xenopus models . This broad species reactivity makes DVL3 antibodies valuable tools for comparative studies across different model organisms .

What are the optimal storage and handling conditions for maintaining DVL3 antibody activity?

For maximum stability and activity retention, DVL3 antibodies should be stored at -20°C in appropriate buffer conditions. The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibodies remain stable for one year after shipment. Notably, for -20°C storage, aliquoting is generally unnecessary for smaller quantities (such as 20μl sizes) that contain 0.1% BSA as a stabilizer . When handling biotin-conjugated antibodies specifically, it's important to avoid repeated freeze-thaw cycles which can compromise the biotin-streptavidin binding activity. Additionally, exposure to strong light should be minimized as biotin conjugates can be photosensitive. Before each use, antibodies should be gently mixed by inversion rather than vortexing to prevent protein denaturation that could affect binding specificity .

What are the recommended dilutions for DVL3 antibodies in different applications?

The optimal dilution of DVL3 antibodies varies significantly depending on the specific application and experimental context. For Western blotting, a dilution range of 1:2000-1:6000 typically provides optimal results . Immunohistochemistry applications generally require a more concentrated antibody preparation, with recommended dilutions ranging from 1:500-1:2000 . For immunofluorescence and immunocytochemistry, dilutions between 1:50-1:500 are suggested . When performing immunoprecipitation, researchers should use 0.5-4.0 μg of antibody for each 1.0-3.0 mg of total protein lysate . For biotin-conjugated DVL3 antibodies specifically, these dilutions may need adjustment as the biotin-streptavidin system provides signal amplification. It is strongly recommended that researchers optimize dilutions for their specific experimental system, as results can be sample-dependent .

How can DVL3 antibodies be employed to study Wnt signaling pathway dynamics?

DVL3 antibodies serve as crucial tools for investigating the temporal-spatial dynamics of Wnt signaling. Research has demonstrated that DVL3-based supermolecular complexes undergo significant size changes in response to Wnt3a stimulation, with complex molecular weights increasing from homodimeric DVL3 to well-defined peaks harboring supermolecular complexes of 0.4 to 2.0 MDa within 30 minutes of Wnt3a addition . To effectively study these dynamics, researchers can combine immunoprecipitation using DVL3 antibodies with size-exclusion chromatography to isolate and characterize these complexes. Fluorescence correlation microscopy using labeled DVL3 antibodies provides another powerful approach for real-time visualization of complex formation in live cells . Additionally, combining DVL3 antibodies with antibodies against other Wnt pathway components (such as β-catenin, Axin, or GSK3β) in co-immunoprecipitation experiments can reveal the dynamic composition of signaling complexes at different time points following Wnt stimulation .

What methodological approaches are optimal for detecting DVL3 phosphorylation states?

The detection of DVL3 phosphorylation states requires sophisticated methodological approaches combining antibody-based techniques with phosphoproteomic methods. Researchers have successfully employed a workflow involving immunoprecipitation of FLAG-tagged DVL3 from transfected HEK293 cells, followed by TiO2 enrichment of phosphorylated peptides and liquid chromatography tandem mass spectrometry . This approach has enabled identification and quantification of DVL3 phosphorylation induced by various kinases. For targeted analysis of specific phosphorylation sites, researchers can use phospho-specific antibodies in combination with standard DVL3 antibodies to determine phosphorylation ratios. Western blotting with phospho-specific antibodies following treatment with kinase inhibitors (such as the CK1ε inhibitor PF-670462 at 10 μM) provides valuable insights into regulatory mechanisms . Additionally, functional characterization of phosphorylation can be achieved through expression of phospho-mimicking/non-phosphorylatable DVL3 mutants combined with FRET assays and NMR spectroscopy to establish structure-function relationships .

How should immunoprecipitation protocols be optimized for studying DVL3 protein complexes?

Optimizing immunoprecipitation protocols for DVL3 protein complexes requires careful consideration of experimental conditions to preserve native interactions. Based on published protocols, cells should be harvested 48 hours post-transfection to allow sufficient protein expression and complex formation . For DVL3 complex isolation, gentle cell lysis conditions are crucial - typically using buffers containing mild detergents (such as 0.5% NP-40 or 1% Triton X-100) rather than harsh ionic detergents that could disrupt protein-protein interactions. When using FLAG-tagged DVL3, anti-FLAG affinity resins provide efficient isolation with minimal background . For endogenous DVL3 complexes, approximately 0.5-4.0 μg of DVL3 antibody should be used per 1.0-3.0 mg of total protein lysate . The addition of phosphatase inhibitors is essential to preserve phosphorylation states, while protease inhibitors prevent degradation during processing. For detecting Wnt-dependent changes in complex formation, cells should be stimulated with Wnt3a for specific time periods (typically 30 minutes) before lysis . Following isolation, complexes can be analyzed by various methods including Western blotting, mass spectrometry, or size-exclusion chromatography to determine their composition and molecular weight .

How can DVL3 antibodies be utilized in reporter assays to study Wnt pathway activation?

DVL3 antibodies can be strategically integrated into reporter assay workflows to correlate pathway activity with protein dynamics. Researchers have successfully combined immunological detection of DVL3 complexes with functional luciferase-based reporter assays to establish mechanistic relationships. A validated approach involves transfecting cells with the canonical Wnt pathway reporter Super8X TopFlash construct (containing Tcf/Lef binding sites) alongside a Renilla luciferase construct for normalization . By simultaneously monitoring DVL3 complex formation (through immunoprecipitation followed by Western blotting) and transcriptional output (via dual-luciferase assay), researchers can directly correlate DVL3 dynamics with pathway activity . For instance, studies have demonstrated that DKK1 treatment effectively blocks both Wnt3a-stimulated Lef/Tcf-sensitive transcriptional responses and the formation of large (>2.0 MDa) supermolecular DVL3-based complexes . Similarly, overexpression of DVL1 and DVL3 has been shown to stimulate both canonical pathway activation and increased formation of very large DVL3-based complexes . These parallel analyses provide powerful insights into the relationship between DVL3 molecular organization and functional outputs of the Wnt signaling pathway.

What critical controls should be included when using DVL3 antibodies in experimental designs?

Rigorous experimental design with DVL3 antibodies requires multiple levels of controls to ensure specificity, sensitivity, and biological relevance. For antibody validation, researchers should incorporate both positive controls (cell lines with confirmed DVL3 expression such as A549, MCF-7, and Raji cells) and negative controls (ideally DVL3-knockout cell lines, such as the documented DVL1/2/3-null HEK293 T-REx cells) . Isotype controls matching the DVL3 antibody class (such as rabbit IgG for polyclonal antibodies or mouse IgG1 kappa for monoclonal antibodies like 4D3) should be included at equivalent concentrations to distinguish specific from non-specific binding . For functional studies, pharmaceutical inhibitors like the CK1ε inhibitor PF-670462 (10 μM) provide valuable controls for pathway modulation . When using biotin-conjugated antibodies specifically, additional controls should include biotin blocking steps and streptavidin-only samples to account for endogenous biotin or non-specific streptavidin binding. For Wnt pathway studies, parallel treatments with pathway activators (Wnt3a) and inhibitors (DKK1) provide critical functional controls, as these have been shown to respectively promote or prevent the formation of DVL3-based supermolecular complexes .

How do experimental approaches differ when studying interactions between DVL3 and other Dishevelled isoforms?

Studying interactions between DVL3 and other Dishevelled isoforms (DVL1, DVL2) requires specialized experimental approaches that differentiate between these highly homologous proteins. Research has demonstrated that these isoforms display both overlapping and distinct functions in Wnt signaling, with DVL3 showing unique complex formation patterns . For effective discrimination, isoform-specific antibodies with confirmed specificity are essential. Co-immunoprecipitation experiments using DVL3 antibodies followed by blotting with isoform-specific antibodies can reveal hetero-oligomerization between different Dishevelled proteins . Knockout models provide another powerful approach, with documented DVL1/2/3-null HEK293 T-REx cells serving as excellent experimental systems for reconstitution experiments . These cells allow systematic expression of individual or combined DVL isoforms to assess their specific contributions to complex formation and signaling. Functional studies have revealed that knockdown of different DVL isoforms produces distinct effects on supermolecular complex formation - while DVL3 knockdown predictably eliminates DVL3 complex detection, DVL1 knockdown specifically attenuates the ability of Wnt3a to promote formation of larger (>2.0 MDa) complexes without affecting basal complex distribution .

What technical considerations apply to visualizing DVL3 punctae formation using immunofluorescence?

Visualizing DVL3 punctae formation via immunofluorescence requires attention to several technical parameters to achieve optimal results. For successful immunofluorescence detection of DVL3, antibodies have been validated at dilutions ranging from 1:50-1:500, with HeLa cells serving as a positive control system . When studying Wnt-induced DVL3 punctae formation, timing is critical - research has shown that membrane-associated protein aggregates form in response to Wnt stimulation within defined timeframes, requiring careful experimental planning . For co-localization studies, combinations of DVL3 antibodies with markers for specific cellular compartments provide valuable spatial context. Advanced imaging approaches such as fluorescence correlation spectroscopy (FCS) offer particular advantages for studying the dynamic behavior of DVL3 punctae in living cells . For specialized applications, researchers have successfully employed engineered DVL3 constructs such as the DVL3 FlAsH III sensor, which can be labeled with FlAsH (500 nM) in the presence of 1,2-ethanedithiol (12.5 μM) for dynamic visualization . This approach requires subsequent washing with 250 μM EDT to reduce non-specific labeling before final washes with HBSS and maintenance in DMEM medium .

What cell models are most appropriate for studying DVL3 function with antibody-based techniques?

Selection of appropriate cell models is crucial for studying DVL3 function using antibody-based techniques. Human embryonic kidney (HEK) cell lines, particularly HEK293T and HEK293 T-REx variants, have been extensively validated and serve as robust models for DVL3 studies . These cells display appropriate responses to Wnt pathway modulators and support efficient transfection for expression of tagged DVL3 variants. For knockout studies, the engineered DVL1/2/3-null HEK293 T-REx cells provide an excellent background for reconstitution experiments with wild-type or mutant DVL3 . For cancer-related research, A549 (lung), MCF-7 (breast), and Raji (lymphoma) cell lines have been confirmed to express detectable levels of endogenous DVL3 . HeLa cells have been validated for immunofluorescence detection of DVL3 and are suitable for subcellular localization studies . Mouse F9 totipotent embryonic carcinoma cells represent another valuable model system, particularly for developmental studies, as they form well-defined DVL3-based complexes that respond dynamically to Wnt3a stimulation . When working with these models, cells should be maintained in DMEM supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics (50 units/ml penicillin, 50 units/ml streptomycin) to ensure optimal growth and experimental consistency .

What potential cross-reactivity issues might arise with DVL3 antibodies and how can they be mitigated?

Cross-reactivity represents a significant concern when working with DVL3 antibodies due to the high sequence homology between Dishevelled family members (DVL1, DVL2, and DVL3). To mitigate these issues, researchers should select antibodies raised against unique regions of DVL3, such as those targeting the C-terminal domain (amino acids 607-704) which shows greater sequence divergence among isoforms . Validation of antibody specificity should include Western blot analysis in systems expressing only specific DVL isoforms, such as DVL1/2/3-null HEK293 T-REx cells reconstituted with individual DVL proteins . For polyclonal antibodies like the rabbit anti-DVL3 (13444-1-AP), antigen affinity purification helps reduce non-specific binding . Optimization of antibody dilution is also critical - excessive antibody concentration increases the risk of cross-reactive binding, so using the minimum effective concentration (e.g., 1:2000-1:6000 for Western blotting) is recommended . When using biotin-conjugated antibodies specifically, additional cross-reactivity can occur with endogenous biotin-binding proteins, requiring appropriate blocking steps with unconjugated streptavidin/avidin before adding the biotin-conjugated primary antibody. Finally, inclusion of appropriate negative controls (isotype-matched non-specific antibodies) and positive controls (isoform-specific overexpression systems) in each experiment provides essential reference points for identifying and quantifying any cross-reactive signals .

How should phosphorylation analysis of DVL3 be designed to identify specific regulatory sites?

Designing phosphorylation analysis of DVL3 requires an integrated approach combining antibody-based detection with mass spectrometry techniques. An effective experimental workflow begins with expression of FLAG-DVL3 in HEK293 cells, with or without co-expression of specific kinases of interest . After cell lysis, DVL3 should be immunoprecipitated using anti-FLAG antibodies, followed by either direct Western blot analysis with phospho-specific antibodies or preparation for mass spectrometry . For comprehensive phosphosite identification, TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry provides the most thorough coverage . When investigating specific kinases, parallel samples should include selective inhibitors - for example, the CK1ε inhibitor PF-670462 at 10 μM has been effectively used to block CK1ε-mediated phosphorylation of DVL3 . For functional characterization, phospho-mimicking (serine/threonine to glutamic acid) and non-phosphorylatable (serine/threonine to alanine) DVL3 mutants should be generated and tested in cellular assays measuring Wnt pathway activation, such as the Super8X TopFlash luciferase reporter system . Additional validation can be provided through biophysical methods such as FRET assays and NMR spectroscopy, which provide structural insights into how phosphorylation affects DVL3 conformation and interaction capabilities .

What are the optimal transfection methods for expressing tagged DVL3 in experimental systems?

The optimization of transfection protocols is essential for successful expression of tagged DVL3 in experimental systems. Polyethylenimine (PEI) transfection has been validated as a cost-effective and efficient method for DVL3 expression in HEK293-derived cell lines . The optimal protocol utilizes PEI at a concentration of 1 μg/ml (pH 7.4) with a PEI:DNA ratio of 6 μl PEI per 1 μg DNA . For standard transfection in 24-well plates, researchers typically use 10 ng of DVL3 expression plasmid combined with reporter constructs (100 ng Super8X TopFlash and 100 ng Renilla luciferase constructs), with the total DNA amount equalized to 400 ng/well using empty vector (pcDNA3.1) . The transfection mixture should be prepared by diluting DNA and PEI separately in plain DMEM (without FBS, L-glutamine, or antibiotics) before combining . For optimal expression, the transfection medium should be replaced with complete DMEM after 6 hours . When planning experiments with DVL3 antibodies, timing is critical - cells should be harvested 24 hours post-transfection for immunoblotting or immunocytofluorescence, while immunoprecipitation experiments require 48 hours of expression to achieve sufficient protein levels . For specialized applications such as FlAsH labeling of DVL3 sensors, additional post-transfection processing steps are required, including washing with HBSS containing 1.8 g/l glucose followed by incubation with FlAsH and EDT .

What strategies can address common troubleshooting issues when using DVL3 antibodies in Western blotting?

Addressing common troubleshooting issues with DVL3 antibodies in Western blotting requires systematic optimization of multiple parameters. For weak or absent signals, optimization of primary antibody concentration is essential - while the recommended range is 1:2000-1:6000, researchers should test a dilution series to determine optimal concentration for their specific sample type . Extended incubation times (overnight at 4°C) often improve signal detection for low-abundance samples. When working with biotin-conjugated DVL3 antibodies specifically, using freshly prepared streptavidin-HRP conjugates can significantly enhance detection sensitivity. For high background issues, increasing blocking stringency (5% BSA or 5% milk in TBST for 1-2 hours) and additional washing steps (5 washes, 5 minutes each with TBST) can substantially improve signal-to-noise ratio. Multiple or unexpected bands may indicate degradation products, which can be addressed by adding additional protease inhibitors during sample preparation, or post-translational modifications, which may require phosphatase treatment to confirm. The observed molecular weight of DVL3 is approximately 78 kDa, but phosphorylated forms may exhibit mobility shifts . For samples with high lipid content, additional centrifugation steps (15,000 × g, 15 minutes) following cell lysis can improve clarity. Finally, for membrane proteins or large complexes (such as the 2.0 MDa DVL3 complexes), extended transfer times or specialized transfer systems may be necessary to ensure complete protein transfer to the membrane .

How can super-resolution microscopy be combined with DVL3 antibodies to study signalosome formation?

Super-resolution microscopy techniques provide powerful approaches for visualizing DVL3 signalosome formation beyond the diffraction limit of conventional microscopy. When combined with appropriately validated DVL3 antibodies, techniques such as Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), or Single Molecule Localization Microscopy (SMLM) can resolve DVL3-containing punctae with resolution approaching 20-50 nm . For biotin-conjugated DVL3 antibodies specifically, detection can be achieved using streptavidin conjugated to photoswitchable fluorophores optimized for super-resolution applications. Sample preparation requires careful optimization - cells should be fixed with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100, with antibody dilutions typically more concentrated (1:50-1:200) than for conventional immunofluorescence . Multi-color imaging combining DVL3 visualization with other Wnt pathway components can reveal spatial organization within signalosomes. Time-resolved super-resolution approaches are particularly valuable, as research has established that DVL3-based complexes undergo significant size changes within 30 minutes of Wnt3a stimulation . These techniques can be complemented by Fluorescence Correlation Spectroscopy (FCS) to add dynamic information about complex diffusion properties and size distributions in living cells . The combination of these advanced imaging approaches with genetic manipulations (such as expression of mutant DVL3 forms) provides unprecedented insights into the structural organization and dynamic behavior of Wnt signalosomes.

What emerging technologies show promise for studying DVL3 post-translational modifications?

Emerging technologies for studying DVL3 post-translational modifications (PTMs) are revolutionizing our understanding of this protein's regulation. Proximity labeling approaches, such as BioID or TurboID fused to DVL3, enable identification of the dynamic DVL3 interactome under different signaling conditions when combined with biotin-based purification and mass spectrometry analysis. For phosphorylation studies specifically, recent advances in targeted phosphoproteomics using parallel reaction monitoring (PRM) allow quantitative tracking of multiple DVL3 phosphosites simultaneously with high sensitivity . Phospho-specific nanobodies, which offer advantages in size and specificity over traditional antibodies, are emerging as valuable tools for detecting specific DVL3 phosphorylation states in live-cell imaging. CRISPR-based approaches for endogenous tagging of DVL3 with split fluorescent proteins or luminescent reporters provide opportunities to monitor DVL3 conformational changes in response to phosphorylation events in physiologically relevant contexts. Additionally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with immunoprecipitation using DVL3 antibodies offers structural insights into how PTMs affect protein conformation and interaction surfaces . These technologies, when combined with traditional antibody-based approaches, provide multi-dimensional information about how phosphorylation and other modifications regulate DVL3 function in Wnt signaling contexts, potentially revealing new therapeutic targets for diseases involving aberrant Wnt pathway activation.

What considerations apply when designing antibody-based screening assays for compounds targeting DVL3-dependent signaling?

Designing antibody-based screening assays for compounds targeting DVL3-dependent signaling requires careful consideration of multiple parameters to ensure specificity, sensitivity, and physiological relevance. High-throughput screening platforms can employ sandwich ELISA or AlphaLISA formats using DVL3 antibodies to detect changes in protein-protein interactions or phosphorylation states in response to compound treatment. For optimal performance, assays should include both primary screens measuring direct DVL3 binding or modification and secondary cellular assays that assess functional consequences on Wnt pathway activation. The Super8X TopFlash luciferase reporter system in DVL1/2/3-null HEK293 T-REx cells reconstituted with DVL3 provides a validated cellular platform for identifying compounds that specifically modulate DVL3-dependent signaling . For more physiologically relevant screening, high-content imaging assays using fluorescently labeled DVL3 antibodies can detect changes in punctae formation and subcellular localization in response to compound treatment . Critical controls should include known Wnt pathway modulators such as DKK1 (which blocks formation of large DVL3-based complexes) and Wnt3a (which promotes their formation) . For biotin-conjugated DVL3 antibodies specifically, assay development should account for potential interference from compounds with biotin-like structures. Compounds identified through these screening approaches have potential applications in various disease contexts where aberrant Wnt signaling contributes to pathology, including cancer, fibrosis, and neurodegenerative disorders.