FZD2 Antibody

What is FZD2 and what is its role in cellular signaling?

FZD2 (Frizzled homolog 2) is a member of the G-protein coupled receptor Fz/Smo family that serves as an important receptor in the Wnt signaling pathway. It contains an FZ domain involved in binding with Wnt ligands . FZD2 is widely expressed in adult tissues including heart, placenta, skeletal muscle, lung, kidney, pancreas, prostate, testis, ovary, and colon . In fetal development, it is expressed in brain, lung, and kidney with low levels in fetal liver . Importantly, FZD2 has been identified as a critical component in both canonical (β-catenin dependent) and non-canonical Wnt signaling pathways that regulate cell migration, invasion, and epithelial-mesenchymal transition (EMT) . Recent research has also shown that FZD2 deficiency increases YAP activity by stabilizing the YAP protein, suggesting its role in regulating the Hippo pathway as well .

What are the common applications for FZD2 antibodies in research?

FZD2 antibodies are versatile tools in multiple experimental applications:

These applications have been validated across human, mouse, and rat samples . The optimal dilutions should be determined for each experimental system to obtain the best results. Most commercially available FZD2 antibodies are rabbit polyclonal antibodies that recognize specific epitopes, including extracellular domains that are accessible in live cell applications .

How should samples be prepared for optimal FZD2 detection in Western blots?

For optimal detection of FZD2 in Western blots, follow these methodological considerations:

• Tissue/Cell Lysis: Use RIPA buffer with protease inhibitors to effectively extract membrane proteins like FZD2. Since FZD2 is a transmembrane protein, ensure complete solubilization.

• Protein Loading: Load 20-40 μg of total protein per lane for cell lysates and 40-60 μg for tissue samples to ensure adequate signal.

• Gel Percentage: Use 8-10% SDS-PAGE gels to achieve good resolution in the 64-70 kDa range where FZD2 is detected .

• Transfer Conditions: Transfer at 100V for 60-90 minutes using 0.45 μm PVDF membrane optimized for larger proteins.

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

• Antibody Incubation: Incubate with primary FZD2 antibody at recommended dilutions (typically 1:500-1:1000) overnight at 4°C .

• Validation Controls: Include a blocking peptide control when first establishing the protocol to confirm antibody specificity, as demonstrated in the validation data from Alomone Labs where preincubation with the Frizzled-2/FZD2 extracellular blocking peptide eliminated the specific band .

What are the best practices for immunofluorescence detection of FZD2?

For effective immunofluorescence (IF) detection of FZD2:

• Cell Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature. For membrane protein preservation, avoid methanol fixation which can disrupt membrane structures.

• Permeabilization: If detecting intracellular domains, permeabilize with 0.1-0.3% Triton X-100 for 5-10 minutes. For extracellular domain detection (using antibodies like AFR-022), permeabilization may be omitted for live cell staining .

• Blocking: Block with 5% normal serum (from the species of secondary antibody) with 1% BSA in PBS for 30-60 minutes.

• Antibody Dilution: Use FZD2 antibodies at 1:50-1:500 dilution depending on the specific antibody and cell type . Start with manufacturer recommendations and optimize as needed.

• Validated Cell Lines: U2OS cells have been confirmed for positive IF/ICC detection with certain FZD2 antibodies .

• Counterstaining: Use DAPI for nuclear staining and consider membrane markers (such as WGA) when assessing membrane localization of FZD2.

• Controls: Include negative controls (secondary antibody only) and positive controls (cell lines with known FZD2 expression) in each experiment.

How can FZD2 antibodies be used to investigate Wnt signaling pathways?

FZD2 antibodies provide valuable tools for investigating both canonical and non-canonical Wnt signaling pathways:

• Co-immunoprecipitation (Co-IP): Use FZD2 antibodies to pull down FZD2 and associated proteins to identify binding partners in the Wnt pathway. This can help distinguish between canonical (β-catenin-dependent) and non-canonical pathways .

• Pathway Inhibition Studies: Apply FZD2-neutralizing antibodies to block receptor function and observe downstream effects on signaling cascades. Research has shown that targeting FZD2 with antibodies can reduce cell migration, invasion, and inhibit tumor growth and metastasis in xenografts .

• EMT Marker Analysis: Monitor changes in epithelial markers (E-cadherin, Occludin) and mesenchymal markers (MMP2, MMP9, Slug) after FZD2 depletion or neutralization to assess EMT regulation .

• Combined Immunofluorescence: Perform double-staining with FZD2 antibodies and other pathway components (β-catenin, YAP, etc.) to visualize co-localization during active signaling.

• Functional Assays: Correlate FZD2 expression levels (detected by immunoblotting) with functional assays such as wound healing (migration), invasion assays, and cell proliferation to establish the biological relevance of FZD2 in your experimental system .

How can FZD2 antibodies be used to study the role of FZD2 in cancer and metastasis?

FZD2 plays a significant role in cancer progression and metastasis, particularly through the regulation of EMT. Research has demonstrated that FZD2 and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors . Here are methodological approaches using FZD2 antibodies:

• Expression Correlation Studies: Use immunohistochemistry with FZD2 antibodies on tissue microarrays to correlate FZD2 expression with tumor grade, stage, and patient outcomes. High levels of FZD2 have been significantly correlated with poor prognosis in breast cancer patients .

• Metastasis Pathway Analysis: Combine FZD2 immunodetection with the assessment of non-canonical signaling components including Fyn and Stat3, which have been identified as part of a previously unrecognized pathway driven by FZD2 .

• Therapeutic Target Validation: Evaluate therapeutic potential by using FZD2 antibodies to neutralize receptor function in xenograft models. Research has shown that antibodies to FZD2 can reduce cell migration and invasion and inhibit tumor growth and metastasis in xenografts .

• EMT Marker Profiles: When FZD2 is depleted, monitor changes in mesenchymal markers (Spp1, Mmp2, Snai2/Slug, Mmp9) and epithelial markers (Cdh1, Ocln, Gsc) using a combination of immunoblotting, qPCR, and immunofluorescence techniques .

• Migration Assays: Quantitatively assess the impact of FZD2 antibody treatment on cancer cell migration using real-time wound-healing assays, which have shown significant reduction in closure time in FZD2-depleted cells compared to control cells .

What are the key considerations when using FZD2 antibodies in knockout/knockdown validation studies?

When using FZD2 antibodies for validation of knockout (KO) or knockdown (KD) systems:

• Antibody Specificity: Ensure the antibody's epitope is removed in your KO model. For example, if using CRISPR to target specific exons, confirm the antibody's binding site is within the deleted region.

• Multiple Antibody Validation: Use multiple antibodies targeting different epitopes of FZD2 to confirm knockout efficiency. The search results indicate several published studies using KD/KO approaches with FZD2 antibody validation .

• Conditional Knockout Systems: For tissue-specific studies, validate the cell-specific loss of FZD2 as demonstrated in the cardiac myocyte-specific knockout model where immunological staining for FZD2 and ACTC1 (cardiac muscle-specific actin) confirmed the loss of FZD2 specifically in cardiomyocytes while maintaining expression in epicardium, endocardium, and valves .

• Functional Validation: Beyond protein detection, validate the functional consequences of FZD2 loss. For example, in FZD2 CKO hearts, increased BrdU incorporation in cardiomyocytes confirmed the functional impact of FZD2 deletion on cell cycle activity .

• Control Cell Selection: When validating FZD2 antibodies in knockout systems, include appropriate positive controls such as heterozygous (HET) models or wild-type cells from the same tissue to account for tissue-specific expression patterns .

How do different FZD2 antibodies compare in detecting specific functional domains of the protein?

Different FZD2 antibodies target distinct epitopes, which affects their utility in specific applications:

When studying FZD2 function:

• Extracellular Domain Antibodies: Antibodies targeting the extracellular N-terminus (such as Alomone's AFR-022) are particularly valuable for:

  • Blocking Wnt ligand interactions in functional studies

  • Flow cytometry analysis of surface expression

  • Live cell imaging applications

• C-terminal Domain Antibodies: These are better suited for:

  • Detecting intracellular signaling complexes

  • Co-immunoprecipitation of downstream effectors

  • Assessing receptor processing and turnover

• Functional Blocking Studies: For investigating non-canonical FZD2 pathways (like the Fyn-Stat3 axis), select antibodies that target the specific domains involved in these interactions .

• Post-translational Modification Detection: Consider whether the antibody's epitope might be affected by glycosylation or phosphorylation, which could impact detection in different experimental contexts.

What are common challenges in detecting FZD2 and how can they be overcome?

Researchers may encounter several challenges when working with FZD2 antibodies:

• Membrane Protein Extraction: As a seven-transmembrane receptor, FZD2 can be difficult to extract efficiently.

  • Solution: Use specialized membrane protein extraction buffers containing adequate detergents (0.5-1% NP-40 or Triton X-100). Consider using RIPA buffer with 0.1% SDS for more complete solubilization.

• Variable Glycosylation: The observed molecular weight of FZD2 (64-70 kDa) may vary due to post-translational modifications .

  • Solution: If precise molecular weight determination is critical, consider deglycosylation treatment with PNGase F before Western blot analysis.

• Cross-reactivity with Other Frizzled Family Members: The Frizzled family contains 10 closely related members with structural similarities.

  • Solution: Use antibodies validated against multiple Frizzled family members and include appropriate positive and negative controls. Consider using knockout or knockdown validation as described in published applications .

• Low Endogenous Expression: Some cell types may express low levels of FZD2, making detection challenging.

  • Solution: Optimize protein loading (up to 60 μg for tissue samples), extend exposure times, and use enhanced chemiluminescence detection systems. Consider concentration steps for immunoprecipitation before Western blotting.

• Background in Immunofluorescence: High background can obscure specific FZD2 staining.

  • Solution: Increase blocking time (up to 2 hours), use higher dilutions of primary antibody with longer incubation times (overnight at 4°C), and include additional washing steps with 0.1% Tween-20 in PBS.

How should contradictory results between different FZD2 antibodies be interpreted?

When faced with contradictory results using different FZD2 antibodies:

• Epitope Accessibility: Different antibodies target different regions of FZD2 which may be differentially accessible depending on:

  • Protein conformation in different signaling states

  • Interaction with binding partners

  • Membrane localization or internalization

  Approach: Map the epitopes of different antibodies and consider whether cellular conditions might affect their accessibility.

• Specificity Validation: Confirm the specificity of each antibody using:

  • Blocking peptide experiments as demonstrated with Alomone's antibody

  • FZD2 knockdown/knockout samples as negative controls

  • Overexpression systems as positive controls

• Application-Specific Optimization: An antibody may perform well in one application but poorly in another.

  • Approach: Optimize protocols specifically for each application (WB, IF, flow cytometry) rather than using the same conditions across applications.

• Isoform Detection: Consider whether antibodies might detect different isoforms or splice variants.

  • Approach: Review the literature and sequence databases for known FZD2 variants and align them with antibody epitopes.

• Contextual Expression: FZD2 expression and modification patterns may vary across tissues and cell types.

  • Approach: When comparing results between different experimental systems, consider tissue-specific factors that might affect antibody performance.

What controls are essential when establishing new experimental protocols with FZD2 antibodies?

When establishing protocols with FZD2 antibodies, these controls are essential:

• Positive Controls: Include samples with known FZD2 expression:

  • Cell lines: Caco-2, HEK-293, U2OS, MDA-231, and THP-1 cells

  • Tissues: Mouse colon, rat kidney

• Negative Controls:

  • Primary antibody omission control to assess secondary antibody specificity

  • FZD2 knockout or knockdown samples when available

  • Cells known to express minimal FZD2 (tissue-specific, may require literature review)

• Blocking Peptide Controls: Preincubate the antibody with its immunizing peptide to confirm signal specificity, as demonstrated in the Alomone Labs validation data .

• Isotype Controls: Include a matched isotype control antibody (e.g., rabbit IgG for rabbit polyclonal FZD2 antibodies) at the same concentration to identify non-specific binding.

• Titration Experiments: Perform antibody dilution series to determine optimal concentration:

  • For Western blot: Test dilutions from 1:200 to 1:2000

  • For immunofluorescence: Test dilutions from 1:50 to 1:500

• Cross-validation: When possible, validate findings using at least two different antibodies against FZD2 that recognize different epitopes.

• Functional Validation: In addition to protein detection, include functional experiments to confirm that observed changes in FZD2 lead to expected downstream effects on Wnt signaling or cell behavior .

How are FZD2 antibodies being used to explore new therapeutic approaches in cancer?

FZD2 antibodies are emerging as valuable tools in cancer research and potential therapeutic development:

What are the methodological considerations when studying FZD2 interactions with other Wnt pathway components?

When investigating FZD2 interactions with Wnt ligands and other pathway components:

• Co-immunoprecipitation (Co-IP) Approach:

  • Use membrane-compatible lysis buffers (containing 0.5-1% NP-40 or digitonin) that maintain protein-protein interactions

  • Consider crosslinking approaches for transient interactions

  • Include appropriate controls (IgG control, reverse Co-IP)

  • Validate interactions with multiple antibodies

• Proximity Ligation Assay (PLA):

  • A powerful technique for visualizing protein-protein interactions in situ

  • Requires validated antibodies from different species against FZD2 and potential interaction partners

  • Provides spatial information about where in the cell these interactions occur

• Bioluminescence Resonance Energy Transfer (BRET)/Förster Resonance Energy Transfer (FRET):

  • For live-cell studies of dynamic interactions

  • Requires construction of fluorescent fusion proteins

  • Can assess conformational changes upon ligand binding

• Surface Plasmon Resonance (SPR):

  • For quantitative binding kinetics between purified FZD2 and Wnt ligands

  • Requires purified protein components or receptor ectodomains

  • Can determine binding affinities and kinetics

• Ligand Considerations:

  • FZD2 interacts with multiple Wnt ligands, particularly Wnt5a/b in the context of EMT and metastasis

  • When studying specific Wnt-FZD2 interactions, consider using recombinant Wnt proteins with tags for detection and pull-down

How can researchers integrate FZD2 antibody-based studies with genomic and transcriptomic approaches?

Integrating FZD2 protein studies with genomic and transcriptomic approaches provides a more comprehensive understanding of FZD2 biology: