FZD1 Antibody, FITC conjugated

What is FZD1 antibody and what biological pathways does it help study?

FZD1 antibody targets Frizzled Family Receptor 1 (FZD1), a crucial receptor in the Wnt signaling pathway. FZD1 functions as a receptor for Wnt proteins and is primarily coupled to the beta-catenin canonical signaling pathway. This pathway leads to the activation of disheveled proteins, inhibition of GSK-3 kinase, nuclear accumulation of beta-catenin, and activation of Wnt target genes . Recent structural studies have demonstrated that FZD1 exhibits constitutive Gq activity, indicating its role in G protein-coupled receptor (GPCR) signaling . The antibody enables researchers to study these pathways by allowing detection, visualization, and quantification of FZD1 expression and distribution in cells and tissues.

How does FITC conjugation enhance the utility of FZD1 antibody?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the FZD1 antibody, eliminating the need for secondary antibody detection systems. This direct labeling approach offers several advantages: reduced experimental time, decreased background signals from secondary antibody cross-reactivity, and compatibility with multi-label immunofluorescence experiments. The bright green fluorescence of FITC (excitation ~495 nm, emission ~519 nm) enables direct visualization of FZD1 in fluorescence microscopy, flow cytometry, and other fluorescence-based detection methods . FITC conjugation particularly enhances applications requiring direct detection of target proteins without additional amplification steps, such as flow cytometry analysis of cell surface receptor expression.

What are the primary applications for FZD1 antibody, FITC conjugated?

The FZD1 antibody, FITC conjugated, has several key applications in research settings:

While ELISA is specifically noted as an application for the commercial product , the FITC conjugation suggests potential utility in other fluorescence-based applications, similar to other FITC-conjugated antibodies used in research contexts .

What controls should be included when using FZD1 antibody, FITC conjugated?

Proper experimental controls are essential for generating reliable data with FZD1 antibody, FITC conjugated:

• Isotype Control: Include a FITC-conjugated rabbit IgG isotype control at the same concentration as the FZD1 antibody to assess non-specific binding .

• Negative Cell Control: Include cells known to express minimal or no FZD1 to establish background fluorescence levels.

• Positive Cell Control: Use cell lines with documented FZD1 expression (based on FZD1's role in Wnt signaling and G protein coupling) .

• Blocking Control: Pre-incubate the antibody with recombinant FZD1 protein (specifically the immunogen used to generate the antibody, amino acids 73-322) before sample application to demonstrate binding specificity.

• Autofluorescence Control: Include an unstained sample to assess natural sample fluorescence in the FITC channel.

These controls help distinguish specific signals from background and validate antibody performance in each experimental context.

How should samples be prepared for optimal results with FZD1 antibody, FITC conjugated?

Sample preparation significantly impacts the performance of FZD1 antibody, FITC conjugated:

For protein detection (ELISA):

• Maintain protein integrity through proper sample collection and storage

• Use a standardized protein extraction protocol with protease inhibitors

• Consider mild fixation methods that preserve protein epitopes

• Perform adequate blocking (typically with BSA or serum) to reduce non-specific binding

For cellular applications:

• For fixed samples, select fixatives that preserve epitope structure (4% paraformaldehyde is often suitable)

• For membrane proteins like FZD1, avoid harsh permeabilization that could disrupt membrane integrity

• Consider native protein conformation when preparing samples, as FZD1 is a seven-transmembrane receptor with critical structural elements

• When studying G protein coupling or other functional aspects, preserve cellular integrity and signaling machinery

Based on the immunogen information (amino acids 73-322 of human FZD1) , ensure that fixation and preparation methods do not obscure this region of the protein.

What factors affect the specificity of FZD1 antibody, FITC conjugated?

Several factors can influence the specificity of FZD1 antibody, FITC conjugated:

• Antibody Source and Generation: This polyclonal antibody was generated in rabbits immunized with recombinant Human Frizzled-1 protein (amino acids 73-322) . Polyclonal antibodies contain a mixture of antibodies recognizing different epitopes, which can offer broader detection but potentially more cross-reactivity than monoclonal alternatives.

• Sequence Homology: The Frizzled family contains ten members (FZD1-10) with conserved domains. Antibody specificity depends on targeting unique regions of FZD1 not shared with other family members. The immunogen (amino acids 73-322) should be evaluated for uniqueness to FZD1.

• Species Cross-Reactivity: The antibody is documented to react with human FZD1 , but potential cross-reactivity with FZD1 from other species should be considered based on sequence conservation.

• FITC Conjugation Chemistry: The conjugation process uses amine-reactive chemistry targeting lysine residues. Excessive conjugation can alter antibody binding properties, while insufficient conjugation results in weak signals.

• Storage and Handling: The recommended storage conditions (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) help maintain antibody integrity and conjugate stability.

To verify specificity, researchers should consider knockout/knockdown validation, competitive binding assays, and cross-validation with alternative FZD1 detection methods.

How can background fluorescence be minimized when using FZD1 antibody, FITC conjugated?

Background fluorescence can significantly impact data quality when using FITC-conjugated antibodies. Strategies to minimize background include:

• Optimize Antibody Concentration: Titrate the antibody to determine the optimal concentration that provides specific signal with minimal background. Starting with the manufacturer's recommended dilution, test serial dilutions to identify the optimal signal-to-noise ratio.

• Extended Blocking: Use 5-10% normal serum or BSA in PBS for 1-2 hours at room temperature to block non-specific binding sites.

• Washing Optimization: Implement additional and extended washing steps with PBS containing 0.05-0.1% Tween-20 to remove unbound antibody.

• Autofluorescence Reduction: For tissues with high autofluorescence, consider:

  • Pre-treatment with 0.1-1% sodium borohydride

  • Incubation with 0.1-0.5% Sudan Black B in 70% ethanol

  • Use of specialized autofluorescence quenching reagents

• Optical Filters: Utilize narrow bandpass filters that precisely match FITC's emission spectrum (~519 nm) to minimize detection of autofluorescence.

• Photobleaching Control: Minimize sample exposure to light during preparation and storage to prevent photobleaching of the FITC conjugate.

These approaches should be systematically evaluated to determine which combination provides optimal results for specific sample types.

How can FZD1 antibody, FITC conjugated be used to study G protein coupling mechanisms?

Recent structural studies have revealed that FZD1 exhibits constitutive Gq activity , making the FZD1 antibody, FITC conjugated, a valuable tool for investigating G protein coupling mechanisms:

• Co-localization Studies: Use the FITC-conjugated FZD1 antibody in combination with antibodies against Gq proteins (differently labeled) to visualize potential co-localization in cells through confocal microscopy. This approach can provide spatial information about receptor-G protein interactions.

• FRET/BRET Applications: While not directly mentioned in the search results, researchers could potentially develop Förster Resonance Energy Transfer (FRET) assays using FITC as a donor fluorophore and an acceptor fluorophore-labeled G protein to study proximity and interaction dynamics.

• Flow Cytometry Analysis: Quantify changes in cell surface FZD1 expression following G protein activation or inhibition to understand receptor trafficking in response to signaling events.

• Structure-Function Studies: Use the antibody to detect expression and localization of FZD1 mutants with altered G protein coupling capabilities. The recent structural data showing that FZD1-Gq complexes can be determined at 3.6 Å resolution provides a framework for designing such mutations.

• Pathway Crosstalk Analysis: Investigate potential crosstalk between the canonical Wnt/β-catenin pathway and G protein signaling by monitoring FZD1 distribution and colocalization with downstream effectors under various stimulation conditions.

These approaches can leverage the FITC-conjugated antibody to gain insights into the molecular mechanisms of FZD1-mediated G protein activation and signaling.

What methodologies combine FZD1 antibody, FITC conjugated with other molecular techniques for comprehensive pathway analysis?

Integrating FZD1 antibody, FITC conjugated, with complementary techniques enables comprehensive analysis of Wnt and G protein signaling pathways:

• Multicolor Flow Cytometry: Combine FITC-conjugated FZD1 antibody with PE or APC-conjugated antibodies against downstream signaling molecules (β-catenin, DVL, GSK3β) to simultaneously measure multiple pathway components at the single-cell level.

• Proximity Ligation Assay (PLA): Use FITC-conjugated FZD1 antibody in conjunction with oligonucleotide-linked secondary antibodies and rolling circle amplification to detect protein-protein interactions within the Wnt signaling complex with high sensitivity.

• ChIP-seq Integration: Correlate FZD1 expression patterns (detected by the FITC-conjugated antibody) with β-catenin chromatin occupancy (determined by ChIP-seq) to link receptor activity to transcriptional outcomes.

• Live Cell Calcium Imaging: Combine FITC-labeled FZD1 detection with calcium-sensitive dyes to monitor G protein-mediated calcium flux in real-time, leveraging FZD1's known coupling to Gq .

• Super-resolution Microscopy: Apply techniques like STORM or PALM using the FITC-conjugated antibody to visualize nanoscale organization of FZD1 in membrane microdomains and its colocalization with signaling partners.

• Phosphoproteomics: Correlate FZD1 expression patterns with global phosphoproteomic changes following Wnt stimulation to map signaling networks downstream of receptor activation.

These integrated approaches provide multidimensional data to decode the complex signaling networks regulated by FZD1.

How should researchers interpret structural data in relation to FZD1 antibody binding?

Recent cryo-EM structures of FZD1 provide valuable context for interpreting antibody binding results :

• Epitope Accessibility: The FZD1 antibody targets amino acids 73-322 , which includes portions of the cysteine-rich domain (CRD). The cryo-EM studies indicate that "No density of CRD is observed because of its flexible connection through a disordered linker with the transmembrane domain" . This flexibility could impact epitope accessibility in different conformational states of the receptor.

• Active vs. Inactive States: The structural studies revealed distinct conformations for active (G protein-coupled) and inactive states of FZD1 . Researchers should consider whether the antibody preferentially recognizes either state, which could bias results toward detecting specific receptor populations.

• G Protein Coupling Interface: The structures show FZD1 coupling to Gq , with interactions at the intracellular face of the receptor. When designing experiments, researchers should consider whether antibody binding might interfere with G protein coupling, particularly in live cell applications.

• Transmembrane Domain Organization: The structural data reveals that "P6.43 kinks away from I7.47 and V7.48 and triggers the movement of TM6 at the cytoplasmic end" during activation. This conformational change could potentially affect antibody access to certain epitopes.

• Receptor Dimerization: If FZD1 forms dimers or higher-order oligomers, antibody binding to one protomer might be influenced by the adjacent protomer's conformation or presence of binding partners.

Understanding these structural considerations helps researchers interpret antibody binding patterns in the context of receptor function and conformation.

What approaches are recommended for quantifying FZD1 expression using FITC-conjugated antibodies?

Quantitative analysis of FZD1 expression using FITC-conjugated antibodies requires standardized approaches:

• Flow Cytometry Quantification:

  • Use calibrated fluorescent beads to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Calculate antibody binding capacity (ABC) to estimate receptor numbers per cell

  • Apply compensation matrices to correct for spectral overlap when using multiple fluorophores

  • Report data as median fluorescence intensity (MFI) rather than mean, especially for non-normally distributed populations

• Microscopy-Based Quantification:

  • Establish standardized exposure settings using calibration slides

  • Implement automated image analysis with consistent thresholding parameters

  • Quantify integrated fluorescence intensity within defined regions of interest

  • Use nucleus-to-cytoplasm ratios or membrane-to-cytoplasm ratios for subcellular distribution analysis

• ELISA Quantification:

  • Generate standard curves using recombinant FZD1 protein

  • Implement four-parameter logistic regression for curve fitting

  • Calculate concentrations from standardized optical density measurements

  • Verify linearity within the working range of the assay

• Statistical Considerations:

  • Apply appropriate normalization to control for technical variation

  • Use non-parametric tests for flow cytometry data (typically not normally distributed)

  • Include sufficient biological replicates (minimum n=3) for statistical power

  • Report effect sizes along with p-values to indicate biological significance

These quantitative approaches provide robust metrics for comparing FZD1 expression across experimental conditions while minimizing technical variability.

How can FZD1 antibody, FITC conjugated contribute to cancer research?

FZD1 antibody, FITC conjugated, offers valuable applications in cancer research, where Wnt signaling dysregulation is implicated in numerous malignancies:

• Receptor Expression Profiling: Quantify FZD1 expression levels across cancer cell lines and patient samples using flow cytometry or immunofluorescence microscopy to correlate expression with clinical outcomes and therapeutic responses.

• Pathway Activation Monitoring: Investigate correlations between FZD1 expression/localization and downstream Wnt signaling activation by combining FITC-labeled FZD1 detection with assessment of β-catenin nuclear translocation or target gene expression.

• Drug Discovery Applications: Screen compounds that modulate FZD1 expression, localization, or function by monitoring changes in FITC signal intensity or distribution following treatment.

• Tumor Heterogeneity Analysis: Apply single-cell analysis using flow cytometry with FITC-conjugated FZD1 antibody to characterize receptor expression heterogeneity within tumors and correlate with stem-like properties or treatment resistance.

• Receptor Trafficking Studies: Track endocytosis and recycling of FZD1 following Wnt stimulation or drug treatment to understand receptor dynamics in cancer cells compared to normal cells.

These applications leverage the direct fluorescent detection capability of the FITC-conjugated antibody to provide insights into FZD1's role in cancer biology and potential therapeutic targeting.

What considerations apply when using FZD1 antibody, FITC conjugated in developmental biology research?

Developmental biology research presents unique considerations when using FZD1 antibody, FITC conjugated:

• Temporal Expression Analysis: Map the dynamic changes in FZD1 expression throughout developmental stages using flow cytometry or microscopy, correlating expression patterns with morphogenetic events.

• Tissue-Specific Distribution: Characterize tissue-specific expression patterns of FZD1 in developing embryos through immunofluorescence microscopy, with special attention to regions where planar cell polarity and Wnt signaling play critical roles.

• Co-expression Studies: Combine FITC-labeled FZD1 detection with markers for specific cell lineages or differentiation states to understand the role of FZD1 in cell fate decisions during development.

• Cross-Species Considerations: When studying model organisms, verify cross-reactivity of the human-reactive antibody with the species of interest through sequence alignment and validation experiments.

• Autofluorescence Challenges: Developmental tissues often exhibit high autofluorescence, requiring rigorous controls and potentially spectral unmixing to distinguish specific FITC signals from background.

• Fixation Optimization: Developmental tissues may require specialized fixation protocols to preserve both morphology and epitope accessibility, especially when studying membrane proteins like FZD1.

These considerations help researchers effectively apply FZD1 antibody, FITC conjugated, to developmental biology questions while addressing the technical challenges specific to this field.