DVL1P1 Antibody, FITC conjugated

What is DVL1P1 and what role does it play in cellular signaling?

DVL1P1 (Dishevelled Segment Polarity Protein 1 Pseudogene 1) is a protein that may play a significant role in the signal transduction pathway mediated by multiple Wnt genes . It is structurally related to the DVL1 protein, which participates in Wnt signaling by binding to the cytoplasmic C-terminus of frizzled family members and transducing the Wnt signal to downstream effectors . DVL1 plays roles in both canonical and non-canonical Wnt signaling pathways, which are critical for embryonic development, tissue homeostasis, and can be dysregulated in various diseases including cancer.

While DVL1 has been extensively characterized, research on DVL1P1 continues to evolve. The protein contains sequence from amino acids 152-173 that serves as an immunogen for antibody production . Understanding DVL1P1's function provides important insights into Wnt signaling regulation and potential therapeutic targets.

What are the technical specifications of commercially available DVL1P1 Antibody, FITC conjugated?

The DVL1P1 Antibody, FITC conjugated, is typically a polyclonal antibody raised in rabbits against a peptide sequence from the human putative segment polarity protein dishevelled homolog DVL1P1 (amino acids 152-173) . The antibody is supplied with the following specifications:

These technical specifications are essential for researchers to evaluate the suitability of the antibody for their specific experimental requirements .

How should DVL1P1 Antibody, FITC conjugated be stored and handled to maintain optimal activity?

Proper storage and handling of DVL1P1 Antibody, FITC conjugated is crucial for maintaining its activity and specificity:

• Storage temperature: Upon receipt, store at -20°C or -80°C . The lower temperature of -80°C is recommended for long-term storage.

• Aliquoting: To minimize freeze-thaw cycles, divide the antibody into small aliquots before freezing, especially if you won't use the entire volume at once.

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as this can lead to denaturation and loss of activity .

• Light sensitivity: As FITC is a fluorophore sensitive to light, the antibody should be protected from prolonged exposure to light during handling and storage to prevent photobleaching.

• Buffer conditions: The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation helps maintain stability during freezing.

• Working dilutions: Prepare working dilutions immediately before use and discard any unused diluted antibody.

• Contamination prevention: Use sterile techniques when handling to prevent microbial contamination.

Proper storage and handling significantly impact experimental outcomes and reproducibility. Researchers should carefully monitor storage conditions to ensure antibody performance remains consistent across experiments.

What are the optimized protocols for using DVL1P1 Antibody, FITC conjugated in fluorescence-based applications?

Optimizing protocols for fluorescence-based applications with DVL1P1 Antibody, FITC conjugated requires careful consideration of several parameters:

For Immunofluorescence Microscopy:

• Fixation method optimization: Compare paraformaldehyde (4%) versus methanol fixation to determine which better preserves epitope recognition while maintaining cellular architecture.

• Permeabilization conditions: Test Triton X-100 (0.1-0.5%), saponin (0.1%), or digitonin (10-50 μg/ml) to identify optimal conditions that allow antibody access while preserving subcellular structures.

• Blocking parameters: Use 5-10% normal serum (from the species of the secondary antibody if used in a detection system) with 1-3% BSA to minimize background signal.

• Antibody dilution optimization: Test serial dilutions (typically starting at 1:50-1:200) to determine the optimal signal-to-noise ratio .

• Counterstaining: DAPI (1 μg/ml) for nuclear visualization can provide context for DVL1P1 localization, which may be important for Wnt signaling studies.

For Flow Cytometry:

• Cell preparation: Single-cell suspensions with viability >90% are essential for reliable results.

• Fixation and permeabilization: For intracellular targets, use commercial fixation/permeabilization kits compatible with FITC fluorescence.

• Antibody titration: Test dilutions from 1:10 to 1:500 to determine optimal staining conditions .

• Controls: Include unstained cells, isotype controls (FITC-conjugated rabbit IgG), and positive controls (cell lines known to express DVL1P1).

• Compensation: If performing multicolor flow cytometry, proper compensation is essential due to FITC spectral overlap with other fluorophores.

The distinct advantage of FITC conjugation is that it eliminates the need for secondary antibody incubation, reducing potential cross-reactivity issues and simplifying protocols.

How can potential cross-reactivity between DVL1P1 and related proteins (such as DVL1, DVL2, DVL3) be addressed in experimental design?

Cross-reactivity between DVL1P1 and related proteins presents a significant challenge for specific detection. Researchers can address this through several methodological approaches:

This systematic approach ensures confidence in experimental results when studying specific family members in the dishevelled protein family.

What are the considerations for using DVL1P1 Antibody, FITC conjugated in multiplex immunofluorescence experiments?

Designing effective multiplex immunofluorescence experiments with DVL1P1 Antibody, FITC conjugated requires careful planning to maximize information while avoiding technical artifacts:

• Spectral compatibility: FITC excites at ~495 nm and emits at ~519 nm. When selecting additional fluorophores, choose those with minimal spectral overlap such as:

  • Texas Red (excitation: ~589 nm, emission: ~615 nm)

  • Cy5 (excitation: ~650 nm, emission: ~670 nm)

  • Pacific Blue (excitation: ~410 nm, emission: ~455 nm)

• Antibody compatibility:

  • Host species considerations: When using multiple primary antibodies, select those raised in different host species to enable specific secondary detection

  • For directly conjugated antibodies like DVL1P1-FITC, ensure other conjugated antibodies don't cross-react with your target proteins

• Staining sequence optimization:

  • Sequential staining protocols may be necessary if antibodies are from the same host species

  • Test order of antibody application to determine if one antibody blocks access to other epitopes

• Signal amplification strategies: For low-abundance targets that might be co-detected with DVL1P1, consider:

  • Tyramide signal amplification (TSA) for other markers

  • Use of quantum dots for brighter, more stable fluorescence

  • Note that DVL1P1-FITC signal cannot be amplified through secondary antibodies due to direct conjugation

• Image acquisition parameters:

  • Use sequential scanning to minimize bleed-through

  • Standardize exposure times and gain settings across experimental conditions

  • Consider spectral unmixing algorithms for closely overlapping fluorophores

• Controls for multiplex experiments:

  • Single-stained controls for each fluorophore

  • Fluorescence minus one (FMO) controls

  • Isotype controls for each conjugated antibody

• DVL1P1-specific considerations:

  • As DVL1P1 may interact with components of the Wnt signaling pathway, co-staining with proteins like β-catenin, GSK3β, or Frizzled receptors may provide functional insights

  • Consider cell cycle markers as Wnt signaling activity can vary throughout the cell cycle

This comprehensive approach enables robust multiplex experiments investigating DVL1P1 in complex cellular contexts.

How can DVL1P1 Antibody, FITC conjugated be utilized in studying Wnt signaling pathway dynamics?

The DVL1P1 Antibody, FITC conjugated provides unique opportunities for investigating Wnt signaling dynamics through various methodological approaches:

• Live-cell imaging of Wnt pathway activation:

  • Transfect cells with fluorescently-tagged Wnt pathway components (e.g., RFP-tagged β-catenin)

  • Permeabilize cells with a gentle detergent to allow antibody entry while maintaining viability

  • Use DVL1P1-FITC to visualize DVL1P1 localization during Wnt stimulation

  • Capture images at defined intervals following Wnt ligand addition (e.g., 0, 15, 30, 60 minutes)

  • Quantify co-localization coefficients between DVL1P1 and other pathway components

• Quantitative analysis of DVL1P1 in signalosome formation:

  • Wnt ligand binding triggers formation of signalosomes at the plasma membrane

  • Use super-resolution microscopy (STORM, PALM) with DVL1P1-FITC to quantify:

    • Signalosome size

    • Protein stoichiometry within signalosomes

    • Temporal dynamics of assembly/disassembly

• Differential response to canonical vs. non-canonical Wnt ligands:

  • Compare DVL1P1 localization following treatment with:

    • Wnt3a (canonical pathway activator)

    • Wnt5a (non-canonical pathway activator)

  • Quantify differences in cytoplasmic vs. membrane-associated DVL1P1-FITC signal

• Phosphorylation state analysis:

  • DVL proteins undergo phosphorylation upon Wnt activation

  • Combine DVL1P1-FITC with phospho-specific antibodies (using a compatible fluorophore)

  • Create a temporal map of phosphorylation events during signaling

• Interaction with downstream effectors:

  • Perform proximity ligation assays (PLA) using DVL1P1-FITC and antibodies against potential interacting partners

  • Quantify interaction events during pathway activation/inhibition

• Pharmacological manipulation:

  • Apply pathway modulators including:

    • GSK3β inhibitors (e.g., CHIR99021)

    • Tankyrase inhibitors (e.g., XAV939)

    • Porcupine inhibitors (e.g., LGK974)

  • Monitor DVL1P1 localization and abundance changes using the FITC-conjugated antibody

  • Correlate these changes with downstream signaling outcomes

This multi-faceted approach leverages the direct FITC conjugation to minimize experimental perturbation while maximizing dynamic information acquisition.

What methodological considerations should be addressed when validating DVL1P1 Antibody, FITC conjugated for specific research applications?

Comprehensive validation of DVL1P1 Antibody, FITC conjugated is essential before implementing it in critical research applications. A systematic validation approach should include:

• Specificity validation:

  • Western blot analysis: Confirm antibody detects a single band of appropriate molecular weight in cell/tissue lysates expressing DVL1P1

  • Immunoprecipitation followed by mass spectrometry: Verify antibody pulls down DVL1P1 without significant off-target proteins

  • Genetic ablation controls: Test antibody in CRISPR/Cas9 knockout or siRNA knockdown systems

  • Recombinant protein detection: Test against purified DVL1P1 and related family members

• Sensitivity assessment:

  • Titration experiments: Determine limit of detection using serial dilutions of recombinant protein

  • Signal-to-noise ratio quantification: Calculate across multiple applications and sample types

  • Comparison with alternative detection methods: qPCR for mRNA expression correlation

• Performance across applications:

  • Application-specific validation: For each intended application (ELISA, flow cytometry, immunofluorescence), perform dedicated validation experiments

  • Sample preparation optimization: Test multiple fixation and permeabilization protocols to maximize signal while preserving epitope accessibility

  • Antibody dilution series: Determine optimal working concentration for each application

• FITC conjugation quality control:

  • Degree of labeling (DOL) determination: Calculate fluorophore-to-protein ratio using spectrophotometric methods

  • Free dye assessment: Confirm absence of unconjugated FITC which could increase background

  • Functional impact assessment: Compare binding efficacy of conjugated vs. unconjugated antibody

• Reproducibility testing:

  • Lot-to-lot consistency: Test multiple antibody lots on standardized samples

  • Inter-laboratory validation: If possible, exchange protocols and samples with collaborating labs

  • Long-term stability assessment: Test antibody performance after storage under recommended conditions for varying time periods

• Context-specific validations:

  • Cell type specificity: Verify performance across relevant cell lines and primary cells

  • Tissue-specific artifacts: Assess background and specificity in tissues of interest

  • Species cross-reactivity: Although labeled for human reactivity, test cross-reactivity with model organism samples if relevant to research

Thorough documentation of these validation steps provides confidence in experimental results and facilitates troubleshooting when unexpected results occur.

How can DVL1P1 Antibody, FITC conjugated be integrated into studies of cancer biology and therapeutic development?

The DVL1P1 Antibody, FITC conjugated offers valuable applications in cancer research, particularly given the importance of Wnt signaling in oncogenesis and therapy resistance:

• Diagnostic and prognostic biomarker exploration:

  • Analyze DVL1P1 expression patterns across cancer subtypes using tissue microarrays

  • Correlate expression levels with clinical outcomes and treatment responses

  • Develop quantitative scoring systems based on fluorescence intensity and subcellular localization patterns

• Integration with anti-FITC CAR-T cell therapy platforms:

  • The FITC conjugation creates opportunities for targeted immunotherapy approaches

  • Anti-FITC CAR T cells can recognize cancer cells bound with FITC-labeled antibodies

  • Research workflow would include:
    a. Validating DVL1P1 surface exposure in cancer cells
    b. Optimizing antibody concentration for CAR-T cell recognition
    c. Assessing cytotoxicity in co-culture systems
    d. Evaluating potential for on-target/off-tumor effects

• Wnt pathway inhibitor screening:

  • Use high-content imaging with DVL1P1-FITC to screen compound libraries

  • Quantify changes in DVL1P1 localization, degradation, or signalosome formation

  • Correlate DVL1P1 dynamics with downstream pathway activation markers

  • Identify compounds that specifically modulate DVL1P1 without affecting related family members

• Resistance mechanism investigations:

  • Compare DVL1P1 expression and localization in therapy-sensitive versus resistant cells

  • Use time-lapse imaging to track DVL1P1-FITC during development of resistance

  • Combine with other pathway markers to identify compensatory signaling networks

• Cancer stem cell (CSC) characterization:

  • Wnt signaling is crucial for maintaining cancer stem cell populations

  • Use DVL1P1-FITC to identify and isolate cells with distinct Wnt signaling states

  • Combine with CSC markers (CD44, CD133, ALDH) in flow cytometry to correlate DVL1P1 levels with stemness

• Translational research applications:

  • Develop image analysis algorithms for quantifying DVL1P1 in clinical samples

  • Standardize reporting methods for pathologists evaluating DVL1P1 expression

  • Create reference datasets for interpreting DVL1P1 alterations in patient samples

The direct FITC conjugation provides advantages for multiparameter analyses in complex tumor microenvironments and enables novel therapeutic approaches leveraging antibody targeting specificity.

What are the key considerations for troubleshooting experiments using DVL1P1 Antibody, FITC conjugated?

When experiments with DVL1P1 Antibody, FITC conjugated yield suboptimal results, a systematic troubleshooting approach is essential:

• No signal or weak signal issues:

  • Antibody integrity: Check for signs of denaturation (precipitation, unusual color)

  • Epitope accessibility: Test alternative fixation methods; some fixatives may mask the epitope

  • Target expression level: Verify DVL1P1 expression in your sample by RT-PCR or Western blot

  • Photobleaching: Minimize exposure to light during all steps; use anti-fade mounting media

  • Working concentration: Test higher antibody concentrations (1:50-1:100 range)

  • Detection system sensitivity: Use more sensitive cameras/detectors or increase exposure time

• High background or non-specific signal:

  • Inadequate blocking: Increase blocking time/concentration or try alternative blocking reagents

  • Autofluorescence: Include unstained controls; use tissues/cells known to have low autofluorescence

  • Fixative-induced fluorescence: Try alternative fixation methods

  • Non-specific binding: Include additional wash steps and increase wash buffer stringency

  • Cross-reactivity: Pre-absorb antibody with related proteins (DVL1, DVL2, DVL3)

  • Over-concentration: Dilute antibody further if background is uniformly high

• Inconsistent results across experiments:

  • Sample variability: Standardize sample collection, processing, and storage

  • Antibody storage issues: Aliquot antibody to avoid repeated freeze-thaw cycles

  • Protocol timing variations: Time each step precisely across experiments

  • Lot-to-lot variation: Request information about lot-specific validation from manufacturer

  • Environmental factors: Control room temperature, humidity, and light exposure

• Application-specific troubleshooting:

  • Flow cytometry issues:

    • Dead cell exclusion: Include viability dye to gate out dead cells

    • Cell clumping: Improve single-cell suspension preparation

    • Compensation problems: Run single-color controls for accurate compensation

  • Immunofluorescence issues:

    • Focus problems: Ensure proper mounting and appropriate coverslip thickness

    • Fading signal: Use anti-fade mounting media and minimize exposure during imaging

    • Uneven staining: Check for air bubbles or incomplete reagent coverage

• Experimental design refinements:

  • Positive control inclusion: Include samples known to express DVL1P1

  • Negative control optimization: Use isotype controls (FITC-conjugated rabbit IgG)

  • Alternative antibody comparison: Test an unconjugated DVL1P1 antibody with secondary detection

• Technical validation tests:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Fluorophore activity test: Verify FITC fluorescence using standard fluorescence measurement

  • Antibody binding verification: Perform ELISA against immunizing peptide

This methodical approach helps isolate the source of experimental problems and guides efficient resolution strategies.