znrf3 Antibody

What is ZNRF3 and why are antibodies against it important for research?

ZNRF3 (Zinc and Ring Finger 3) is a type 1 transmembrane E3 ubiquitin ligase that functions as a critical negative regulator of the Wnt signaling pathway. It mediates the ubiquitination and subsequent degradation of Wnt receptor components including Frizzled receptors and LRP6 . ZNRF3 acts as a tumor suppressor in the intestinal stem cell zone by inhibiting Wnt signaling, thereby restricting the size of the intestinal stem cell zone .

Antibodies against ZNRF3 are essential research tools because they allow scientists to:

• Track ZNRF3 expression levels in different tissues and cell types

• Study the subcellular localization of ZNRF3

• Investigate ZNRF3's role in Wnt signaling regulation

• Examine its interactions with other proteins like R-spondin and LGR4/5/6

• Assess its involvement in cancer development and progression

These antibodies help elucidate the fundamental mechanisms of Wnt pathway regulation and may eventually contribute to therapeutic approaches targeting this pathway in cancer and other diseases.

What applications are ZNRF3 antibodies typically validated for?

Based on commercially available options, ZNRF3 antibodies are validated for several applications:

When selecting a ZNRF3 antibody, researchers should verify that it has been validated for their specific application and target species. Most commercial antibodies show reactivity with human ZNRF3, while some are also validated for mouse and rat orthologs .

How can researchers validate the specificity of ZNRF3 antibodies?

To ensure experimental reliability, ZNRF3 antibody specificity should be validated through:

• Positive and negative controls:

  • Use ZNRF3 overexpression systems (e.g., HEK293T cells transfected with ZNRF3 expression vectors)

  • Use ZNRF3 knockdown/knockout systems (e.g., siRNA-treated cells)

• Cross-reactivity testing:

  • Test against the homologous protein RNF43 to ensure specificity

  • Verify minimal reactivity with other RING-domain containing proteins

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide to block specific binding

  • Compare staining patterns with and without peptide blocking

• Multiple antibody validation:

  • Use antibodies raised against different epitopes of ZNRF3

  • Compare staining patterns to confirm consistent results

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that the immunoprecipitated protein is indeed ZNRF3

The search results indicate that many commercial antibodies are raised against synthetic peptides derived from human ZNRF3 , which should be considered when interpreting results, particularly in non-human models.

How can ZNRF3 antibodies be used to study the Wnt signaling pathway?

ZNRF3 antibodies provide valuable tools for investigating Wnt signaling regulation:

• Co-immunoprecipitation studies:

  • Use ZNRF3 antibodies to pull down protein complexes and identify binding partners in the Wnt pathway

  • Detect interactions with Frizzled receptors, LRP6, R-spondins, and LGR4/5/6 proteins

• Dual immunofluorescence:

  • Co-stain with ZNRF3 antibodies and antibodies against other Wnt pathway components

  • Assess colocalization at the cell membrane or in intracellular compartments

• Flow cytometry:

  • Monitor cell surface levels of ZNRF3 and Wnt receptors simultaneously

  • Quantify changes in receptor abundance upon pathway stimulation

• Wnt pathway activation assays:

  • Use ZNRF3 antibodies alongside Topflash reporter assays

  • Correlate ZNRF3 levels with Wnt signaling output

• Phosphorylation studies:

  • Combine ZNRF3 immunoprecipitation with phospho-tyrosine antibodies

  • Investigate how phosphorylation regulates ZNRF3 function in the Wnt pathway

For example, researchers have demonstrated that PTPRK depletion increases tyrosine phosphorylation of ZNRF3, which affects its ability to internalize and degrade Wnt receptors . This finding illustrates how ZNRF3 antibodies can help elucidate regulatory mechanisms within the Wnt pathway.

What methods are most effective for studying ZNRF3 phosphorylation using antibodies?

ZNRF3 is subject to tyrosine phosphorylation, which appears to regulate its function. To study this:

• Immunoprecipitation followed by phospho-tyrosine detection:

  • Immunoprecipitate ZNRF3 using specific antibodies

  • Probe with anti-phosphotyrosine antibodies to detect phosphorylation status

  • Use phosphatase inhibitors like Na-pervanadate to preserve phosphorylation

• Bafilomycin treatment:

  • Inhibit endocytic traffic and lysosomal degradation with bafilomycin

  • This approach has been shown to enhance detection of phosphorylated ZNRF3

• Enzyme-substrate elution assays:

  • Immobilize PTPRK (a phosphatase that acts on ZNRF3)

  • Incubate with cell lysates containing ZNRF3

  • Elute bound ZNRF3 with vanadate and analyze by Western blot

• Quantification methods:

  • Normalize phospho-ZNRF3 signals to total ZNRF3 levels

  • Calculate ratios to determine relative phosphorylation states under different conditions

The research indicates that ZNRF3 phosphorylation is dynamic and often difficult to detect without special measures, as it appears to be rapidly dephosphorylated by phosphatases like PTPRK . Therefore, phosphatase inhibition is often necessary for effective detection.

How can researchers investigate ZNRF3's role in ubiquitination and receptor degradation?

To study ZNRF3's E3 ubiquitin ligase activity and its effects on receptor degradation:

• In vitro ubiquitination assays:

  • Immunoprecipitate ZNRF3 from cell lysates

  • Incubate with recombinant E1, E2, ATP and ubiquitin

  • Detect ubiquitinated products by Western blot

• Receptor degradation assays:

  • Co-express ZNRF3 with tagged Frizzled receptors or LRP6

  • Monitor receptor levels by Western blot

  • Compare wild-type ZNRF3 with RING domain mutants (ZNRF3-ΔRING)

• Cell surface biotinylation:

  • Label surface proteins with biotin

  • Isolate biotinylated proteins using streptavidin

  • Detect ZNRF3 and Wnt receptors by Western blot

• Flow cytometry for receptor quantification:

  • Use pan-FZD antibodies to measure Frizzled levels at the cell surface

  • Compare receptor levels with and without ZNRF3 manipulation

• Lysosomal inhibition studies:

  • Treat cells with lysosomal inhibitors like bafilomycin

  • Assess accumulation of ZNRF3 and its substrates

Research shows that the RING domain of ZNRF3 is essential for its ability to mediate receptor internalization and degradation. ZNRF3-ΔRING is more stable at the plasma membrane compared to wild-type ZNRF3, suggesting that the RING domain is required for ZNRF3's own turnover as well .

What are the key considerations when selecting ZNRF3 antibodies for different experimental approaches?

When choosing ZNRF3 antibodies, researchers should consider:

• Epitope location:

  • Antibodies targeting the extracellular domain are preferred for detecting surface ZNRF3

  • Antibodies against the cytoplasmic domain may be better for detecting total cellular ZNRF3

  • The search results show antibodies targeting different regions (e.g., aa 101-200/936, aa 678-789)

• Species reactivity:

  • Ensure cross-reactivity with your experimental model organism

  • Many antibodies are validated for human, mouse, and rat ZNRF3

• Clonality:

  • Polyclonal antibodies offer multivalent binding and stronger signals

  • Monoclonal antibodies provide higher specificity and reproducibility

  • Both types are commercially available for ZNRF3

• Tag compatibility:

  • When using tagged ZNRF3 constructs (e.g., ZNRF3-HA), ensure antibodies don't interfere with the tag

  • Consider anti-tag antibodies as alternatives in recombinant systems

• Application-specific optimization:

  • For IHC: Consider fixation methods and antigen retrieval requirements

  • For IF: Optimize permeabilization conditions

  • For WB: Determine appropriate protein denaturation conditions

The search results indicate successful use of doxycycline-inducible ZNRF3-HA expression systems when working in cell lines with poor transfection efficiency or when facing a lack of reliable ZNRF3 antibodies .

How can researchers effectively study ZNRF3 trafficking and localization?

To investigate ZNRF3 trafficking and subcellular localization:

• Colocalization with organelle markers:

  • Use dual immunofluorescence with antibodies against:

    • LAMP1 (lysosomal marker)

    • Rab11 (recycling endosome marker)

    • Other endosomal markers (EEA1, Rab5, Rab7)

• Live-cell imaging approaches:

  • Generate fluorescent protein-tagged ZNRF3 constructs

  • Combine with fluorescently labeled R-spondin or Wnt ligands

  • Track trafficking dynamics in real-time

• Density gradient fractionation:

  • Separate cellular components based on density

  • Detect ZNRF3 in different fractions using antibodies

  • Correlate with markers of specific organelles

• Internalization assays:

  • Surface-label ZNRF3 with antibodies at 4°C

  • Allow internalization at 37°C

  • Measure remaining surface signal versus internalized signal

• Proximity labeling approaches:

  • Fuse ZNRF3 to BioID or APEX2

  • Identify proximal proteins in different subcellular compartments

  • Validate interactions with antibody-based methods

Research has shown that ZNRF3 localizes to various intracellular compartments, including lysosomes and recycling endosomes. Additionally, PTPRK knockdown has been demonstrated to increase ZNRF3 surface levels, suggesting a role for PTPRK in promoting ZNRF3 internalization .

What controls should be included when using ZNRF3 antibodies in complex formation studies?

When studying ZNRF3 interactions with proteins like R-spondin and LGR4/5/6:

• Essential negative controls:

  • Isotype control antibodies for immunoprecipitation

  • ZNRF3 knockout or knockdown samples

  • Competitive blocking with immunizing peptides

• Protein interaction controls:

  • Use ZNRF3 mutants lacking key domains (e.g., ZNRF3-ΔRING)

  • Include known non-interacting proteins as negative controls

  • Use established interaction partners as positive controls

• Binding verification approaches:

  • Reverse co-immunoprecipitation (pull down with partner protein antibody)

  • Proximity ligation assays to detect interactions in situ

  • Bio-layer interferometry (BLI) with purified components

• Functional validation:

  • Confirm biological relevance of interactions using reporter assays

  • Assess effects of disrupting interactions on Wnt signaling

  • Measure receptor degradation as a functional outcome

• Structural context:

  • Consider the structural organization of complexes (e.g., LGR4-RSPO2-ZNRF3)

  • Verify proper protein folding and domain accessibility

Research data shows that recombinant human ZNRF3 protein can bind to human R-Spondin 3 with high affinity (10.9-16.05 nM), which serves as a useful positive control for interaction studies .

How can researchers resolve issues with non-specific binding of ZNRF3 antibodies?

When experiencing high background or non-specific binding:

• Optimization strategies:

  • Titrate antibody concentrations to find optimal signal-to-noise ratio

  • Increase blocking stringency (longer blocks, different blocking agents)

  • Optimize washing steps (more washes, higher salt concentration)

  • Pre-adsorb antibodies with tissues/cells lacking ZNRF3

• Cross-reactivity assessment:

  • Test on ZNRF3 knockout samples to identify non-specific bands

  • Compare with antibodies targeting different ZNRF3 epitopes

  • Perform peptide competition assays to identify specific signals

• Sample preparation considerations:

  • For membrane proteins like ZNRF3, optimize detergent types and concentrations

  • Consider native versus denaturing conditions based on application

  • Use appropriate protease and phosphatase inhibitors

• Alternative detection methods:

  • If direct antibody labeling causes issues, consider secondary-only controls

  • Try biotin-streptavidin amplification for weak signals

  • Consider tyramide signal amplification for IHC/IF applications

• Affinity purification:

  • If polyclonal antibodies show high background, consider affinity purification

  • Use immobilized antigen to select for high-affinity antibodies

Commercial ZNRF3 antibodies are often purified by protein A or peptide affinity chromatography to improve specificity , but additional optimization may be necessary for challenging applications.

What are the best methods to distinguish between ZNRF3 and its homolog RNF43 in experimental systems?

ZNRF3 and RNF43 are homologous E3 ubiquitin ligases with overlapping functions, making their distinction important:

• Antibody selection approaches:

  • Choose antibodies targeting non-conserved regions between ZNRF3 and RNF43

  • Validate specificity using overexpression systems for each protein

  • Test for cross-reactivity with both recombinant proteins

• Genetic manipulation strategies:

  • Use specific siRNAs targeting unique regions of each transcript

  • Generate gene-specific knockouts using CRISPR/Cas9

  • Employ rescue experiments with specifically tagged versions

• Functional discrimination methods:

  • Exploit subtle differences in substrate specificity

  • Investigate different regulatory mechanisms (e.g., ZNRF3 regulation by PTPRK)

  • Assess differential expression patterns across tissues

• Combined depletion approaches:

  • Use combined knockdown (si ZNRF3/RNF43) as a control

  • Compare phenotypes between single and double knockdowns

  • This approach has been used to study their collective role in Wnt signaling

• Mass spectrometry verification:

  • Use immunoprecipitation followed by mass spectrometry

  • Identify unique peptides to confirm antibody specificity

Research shows that both ZNRF3 and RNF43 are co-expressed on the cell surface and collectively regulate Wnt signaling through the ubiquitination of LRP6 and Frizzled receptors . Distinguishing their individual contributions requires careful experimental design.

How can ZNRF3 antibodies be utilized in therapeutic development research?

ZNRF3 antibodies have emerging applications in therapeutic development:

• PROTAB development:

  • ZNRF3 shows promise as a potent degrader of disease-causing cell-surface proteins

  • Anti-ZNRF3-antibody-based PROTABs (PROteolysis-TArgeting Bifunctional molecules) are being developed

  • These utilize ZNRF3's E3 ligase activity to target "undruggable" proteins

• Cancer biomarker research:

  • ZNRF3 expression is down-regulated in gastric carcinomas

  • ZNRF3 mutations are linked to carcinomas of the gastric tract, pancreas, liver, and ovary

  • Antibodies can help assess ZNRF3 status in tumor samples

• Wnt pathway modulation:

  • Target the ZNRF3-R-spondin interaction

  • Develop antibodies that enhance or inhibit ZNRF3's ability to suppress Wnt signaling

  • Screen for antibodies that affect ZNRF3 dimerization, which appears important for activity

• Structure-guided approaches:

  • Use antibodies to study the "finger-crossed" arrangement of ZNRF3 TM helices

  • Investigate the dimerization of RING domains, which is critical for E3 ligase activity

  • Leverage structural insights to design more effective therapeutic antibodies

• Regenerative medicine applications:

  • Study ZNRF3's role in intestinal stem cell regulation

  • Develop antibody tools to modulate stem cell proliferation

  • Target the ZNRF3-regulated Wnt pathway for tissue regeneration

Recent structural studies of LGR4-RSPO2-ZNRF3 complexes provide valuable insights that can guide the optimization of PROTABs based on anti-ZNRF3 antibodies, potentially expanding the range of targetable proteins in therapeutic development .

What techniques can help resolve contradictory results when using different ZNRF3 antibodies?

When faced with inconsistent results from different ZNRF3 antibodies:

• Comprehensive epitope mapping:

  • Determine the exact binding sites of each antibody

  • Assess if different epitopes may be differentially accessible in various contexts

  • Use deletion mutants to verify epitope regions

• Post-translational modification analysis:

  • Check if antibodies recognize regions subject to phosphorylation, ubiquitination, or other modifications

  • Research indicates ZNRF3 is tyrosine-phosphorylated, which could affect antibody binding

  • Test antibody recognition under conditions that alter modification status

• Conformational considerations:

  • ZNRF3 adopts different conformations during regulation

  • Some antibodies may preferentially recognize specific conformational states

  • Test under conditions that stabilize different conformations

• Orthogonal validation approaches:

  • Implement non-antibody methods to verify results (e.g., mass spectrometry)

  • Use genetic approaches (mRNA quantification, CRISPR editing)

  • Employ tagged versions of ZNRF3 when possible

• Systematic comparison:

  • Create a standardized testing panel for multiple antibodies

  • Document key parameters (concentration, incubation time, buffer composition)

  • Publish comprehensive validation data to benefit the research community

The complexity of ZNRF3 biology, including its dimerization, different cellular localizations, and dynamic regulation, may contribute to discrepancies between antibodies that recognize different forms or states of the protein .

How can researchers use ZNRF3 antibodies to investigate its role in cancer development?

ZNRF3 functions as a tumor suppressor, making it an important target for cancer research:

• Expression correlation studies:

  • Use ZNRF3 antibodies for IHC analysis of tumor tissue microarrays

  • Correlate ZNRF3 expression with clinical outcomes

  • Compare expression between tumor and adjacent normal tissues

• Mutation impact assessment:

  • Generate antibodies specific to common ZNRF3 mutants

  • Compare localization and function of wild-type versus mutant ZNRF3

  • Investigate how mutations affect interactions with Wnt pathway components

• Signaling pathway analysis:

  • Use phospho-specific antibodies to study ZNRF3 regulation

  • Investigate how oncogenic signals modify ZNRF3 function

  • Examine cross-talk between ZNRF3 and other cancer-related pathways

• Therapeutic response prediction:

  • Determine if ZNRF3 status correlates with response to Wnt pathway inhibitors

  • Develop companion diagnostics using ZNRF3 antibodies

  • Stratify patients based on ZNRF3 expression or mutation status

• Mechanistic studies in model systems:

  • Implement ZNRF3 antibodies in patient-derived xenograft models

  • Track ZNRF3 dynamics during cancer progression

  • Validate findings in organoid systems

Research indicates that ZNRF3 expression is down-regulated in gastric carcinomas, and ZNRF3 mutations are linked to carcinomas of the gastric tract, pancreas, liver, and ovary , suggesting critical roles in multiple cancer types.

What novel techniques are emerging for studying ZNRF3 complexes using antibodies?

Cutting-edge approaches for investigating ZNRF3 complexes include:

• Cryo-electron microscopy:

  • Visualize ZNRF3 complexes with R-spondin and LGR proteins

  • Use antibodies to stabilize complexes for structural determination

  • Recent structural studies have revealed the assembly mechanism of LGR4-RSPO2-ZNRF3 complexes

• Single-molecule imaging:

  • Track individual ZNRF3 molecules in live cells

  • Monitor complex formation and dissociation kinetics

  • Use fluorescently labeled antibody fragments for minimal interference

• Proximity-dependent labeling:

  • Fuse ZNRF3 to BioID or APEX2

  • Map the temporal dynamics of the ZNRF3 interactome

  • Validate interactions using targeted antibody approaches

• Organoid-based studies:

  • Implement ZNRF3 antibodies in 3D intestinal organoid systems

  • Study ZNRF3 function in a physiologically relevant context

  • Examine dynamic regulation in stem cell compartments

• AlphaFold-guided antibody development:

  • Use structural predictions to design antibodies against specific conformations

  • Target functionally important interfaces

  • Structural studies are leveraging AlphaFold predictions to gain insights into ZNRF3 complexes

• NanoBiT complementation assays:

  • LgBiT and HiBiT fusion constructs with LGR4 and ZNRF3 have been used

  • These allow real-time monitoring of protein interactions

  • Can be combined with antibody-based approaches for validation