did2 Antibody

What is DVL2 and why is it important in research?

DVL2 is a segment polarity protein dishevelled homolog encoded by the DVL2 gene in humans. The protein has a molecular weight of approximately 78.9 kilodaltons and plays a crucial role in Wnt signaling pathways . DVL2 acts as a mediator between cell surface receptors and downstream signaling components, making it an important target for studying development, cell polarization, and various disease states including cancer.

What applications are DVL2 antibodies commonly used for?

DVL2 antibodies are frequently employed in multiple research applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunocytochemistry (ICC), and Enzyme-Linked Immunosorbent Assay (ELISA) . Each application requires specific optimization, with Western blotting being particularly valuable for quantifying DVL2 protein levels and identifying potential post-translational modifications.

What species reactivity should I consider when selecting a DVL2 antibody?

When selecting a DVL2 antibody, consider that many commercially available antibodies react with human samples, while some also cross-react with mouse, rat, canine, porcine, and monkey orthologs . For comparative studies across species, it's essential to verify the cross-reactivity of your chosen antibody through literature or validation data from suppliers.

How should I design positive and negative controls for DVL2 antibody experiments?

For robust experimental design, incorporate positive controls using tissues or cell lines known to express DVL2 (such as neuronal tissues or Wnt-responsive cell lines). For negative controls, consider using:

• Primary antibody omission controls

• Isotype controls matching the DVL2 antibody host species

• Blocking peptide competition assays

• Tissue from DVL2 knockout models (where available)

These controls help distinguish specific from non-specific binding and validate antibody performance across experimental conditions .

What variables need to be controlled in DVL2 detection protocols?

When designing experiments for DVL2 detection, systematically control:

Independent Variables:

• Antibody concentration

• Incubation time and temperature

• Blocking reagents

• Sample preparation methods

Dependent Variables:

• Signal intensity

• Signal-to-noise ratio

• Reproducibility across replicates

Extraneous Variables:

• Sample storage conditions

• Potential cross-reactivity with other Dishevelled family members

• Batch-to-batch antibody variation

How can I optimize antibody dilutions for different DVL2 detection applications?

Optimal antibody dilution varies by application. Based on available data, recommended starting dilutions are:

Always titrate the antibody in your specific experimental system to determine optimal conditions .

How can I validate DVL2 antibody specificity for my experimental system?

Validating DVL2 antibody specificity requires a multi-faceted approach:

• Molecular weight verification: Confirm the observed band matches the expected 78.9 kDa size for DVL2

• Knockdown/knockout validation: Use siRNA, CRISPR, or other genetic approaches to reduce DVL2 expression and confirm corresponding reduction in antibody signal

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Orthogonal detection methods: Compare results using antibodies targeting different DVL2 epitopes

• Mass spectrometry validation: For ultimate confirmation, perform immunoprecipitation followed by mass spectrometry

This comprehensive validation approach ensures reliable experimental outcomes and addresses potential non-specific binding issues .

What are the optimal sample preparation methods for DVL2 detection?

Sample preparation significantly impacts DVL2 antibody performance. For optimal results:

For Western Blot:

• Use RIPA or NP-40 based lysis buffers containing protease inhibitors

• Include phosphatase inhibitors if studying DVL2 phosphorylation status

• Avoid repeated freeze-thaw cycles

• Consider subcellular fractionation to separate cytoplasmic from nuclear DVL2 pools

For IHC/IF:

• Test both formalin-fixed paraffin-embedded (FFPE) and frozen sections

• For FFPE, antigen retrieval with TE buffer pH 9.0 often works well (similar to protocols for other membrane-associated proteins)

• For cultured cells, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization typically yields good results

These preparation methods preserve DVL2 antigenicity while maintaining tissue/cellular morphology.

How can I address inconsistent DVL2 signal in Western blot applications?

Inconsistent DVL2 Western blot signals may result from several factors:

• Sample degradation: Ensure complete protease inhibition during sample preparation

• Insufficient transfer: Optimize transfer conditions for the 78.9 kDa DVL2 protein

• Antibody degradation: Aliquot antibodies to avoid repeated freeze-thaw cycles

• Post-translational modifications: DVL2 undergoes phosphorylation and ubiquitination, which can affect migration patterns

• Background issues: Use 5% BSA instead of milk for blocking when significant background appears

Systematic troubleshooting of each variable can resolve inconsistent results. Document optimization steps carefully for reproducible outcomes .

What approaches can resolve contradictory findings between different DVL2 antibodies?

When different DVL2 antibodies yield contradictory results:

• Compare epitope locations: Different antibodies may target distinct regions of DVL2 that are differentially accessible in certain contexts

• Evaluate isoform specificity: Confirm whether antibodies recognize all or specific DVL2 isoforms

• Assess post-translational modifications: Some antibodies may be sensitive to phosphorylation or other modifications

• Check for cross-reactivity: Particularly with other Dishevelled family members (DVL1, DVL3)

• Validate with orthogonal methods: Confirm protein expression using non-antibody methods (e.g., mass spectrometry, RNA expression)

How can DVL2 antibodies be utilized in studying Wnt signaling complexes?

DVL2 antibodies are powerful tools for investigating Wnt signaling complexes through:

• Co-immunoprecipitation: Pull down DVL2 to identify interaction partners in different contexts

• Proximity ligation assays: Visualize in situ protein-protein interactions between DVL2 and other Wnt pathway components

• ChIP-seq applications: Study DVL2 association with chromatin when using nuclear fractions

• FRET/BRET approaches: Combine antibody-based detection with fluorescent protein tags to study dynamic interactions

• Super-resolution microscopy: Investigate DVL2 clustering and membrane association during Wnt pathway activation

These approaches have revealed critical insights into DVL2's role as a scaffolding protein in signal transduction .

What considerations are important when studying DVL2 in relation to other Dishevelled family proteins?

When investigating DVL2 alongside other Dishevelled family members:

• Specificity verification: Confirm antibody specificity against DVL1, DVL2, and DVL3 through overexpression systems

• Functional redundancy: Design experiments acknowledging that Dishevelled proteins have partially overlapping functions

• Tissue-specific expression: Account for differential expression patterns across tissues and developmental stages

• Domain-specific antibodies: Consider using antibodies targeting unique regions outside the conserved DIX, PDZ, and DEP domains

• Compensation mechanisms: In knockdown studies, monitor potential compensatory upregulation of other family members

These considerations help distinguish DVL2-specific roles from general Dishevelled functions in developmental and disease contexts .