WWC2 Antibody

What is WWC2 protein and why is it important in research?

WWC2 (WW and C2 domain containing 2), also known as BOMB (BH3-only member B), is a 1192 amino acid protein with a molecular mass of approximately 133.9 kDa in humans. The protein contains one C2 domain and two WW domains, which are essential for protein-protein interactions . WWC2 has gained research significance due to its involvement in:

• Regulation of the Hippo signaling pathway, which controls organ size and cell proliferation

• Modulation of GABAA receptor-mediated synaptic transmission

• Potential tumor suppressor activity in hepatocellular carcinoma (HCC)

• Cell migration and transcriptional regulation processes

Understanding WWC2's role in these biological processes has implications for neurological disorders and cancer research, making WWC2 antibodies valuable tools for investigating these pathways .

What applications are WWC2 antibodies most commonly used for?

WWC2 antibodies have been validated for multiple research applications with varying effectiveness:

When designing experiments, researchers should note that optimal dilutions are antibody-specific and may require titration for each experimental system to achieve optimal signal-to-noise ratios .

How do I select the appropriate WWC2 antibody for my experiment?

Selection of an appropriate WWC2 antibody should be guided by:

• Target species: Determine if the antibody recognizes WWC2 in your model system (human, mouse, rat). Cross-reactivity information is typically provided in product datasheets .

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, IP). Not all antibodies perform equally across all applications .

• Epitope recognition: Consider which region of WWC2 the antibody recognizes. Different epitopes may be masked or exposed depending on protein conformation or isoform expression .

• Clonality:

  • Monoclonal antibodies (like WWC2 Antibody H-1) offer high specificity to a single epitope

  • Polyclonal antibodies recognize multiple epitopes and may provide stronger signals but with potentially higher background

• Supporting validation data: Review published literature or manufacturer validation data showing the antibody's performance in your application of interest .

What are the optimal conditions for detecting WWC2 using Western blotting?

For optimal Western blot detection of WWC2:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • WWC2 is primarily detected in cytosol and membrane/crude synaptosome fractions, but is depleted from purified excitatory postsynaptic density (PSD)

• Protein loading and separation:

  • Load 20-30 μg of total protein per lane

  • Use 8-10% SDS-PAGE gels due to WWC2's high molecular weight (observed at approximately 150 kDa)

• Transfer conditions:

  • For large proteins like WWC2, use low methanol (5-10%) transfer buffers

  • Consider longer transfer times or lower voltage for complete transfer

• Antibody conditions:

  • Primary antibody: Dilute 1:1000-1:6000 in blocking buffer

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: HRP-conjugated anti-species IgG at 1:5000-1:10000

• Expected results:

  • WWC2 typically appears at approximately 150 kDa

  • Multiple bands may indicate detection of different isoforms (up to 7 reported)

Researchers should validate specificity using positive controls (brain tissue samples) and negative controls (knockdown or knockout samples if available) .

How can I visualize WWC2 subcellular localization using immunofluorescence?

For successful immunofluorescence detection of WWC2:

• Sample preparation:

  • For cultured cells: Fix with 4% paraformaldehyde (10-15 minutes) followed by permeabilization with 0.1-0.2% Triton X-100

  • For tissue sections: Use antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS for 1 hour at room temperature

  • Dilute WWC2 antibodies 1:20-1:200 in blocking buffer

  • Incubate overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies at 1:200-1:500

• Expected localization patterns:

  • WWC2 shows cytoplasmic distribution with potential association with cytoskeletal elements (microtubules)

  • In neurons, WWC2 interacts with inhibitory postsynaptic scaffolds like gephyrin

• Controls and counterstaining:

  • Include DAPI nuclear counterstain for orientation

  • Consider co-staining with markers for subcellular compartments of interest (e.g., gephyrin for inhibitory synapses)

Immunofluorescence has been successfully used to demonstrate altered surface expression of GABAA receptor subunits in WWC2 conditional knockout mice, highlighting the utility of this approach for studying WWC2 function .

How can WWC2 antibodies be used to investigate protein-protein interactions within the Hippo signaling pathway?

WWC2 antibodies are valuable tools for investigating protein-protein interactions in the Hippo pathway through multiple approaches:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4.0 μg of WWC2 antibody per 1-3 mg of total protein lysate

  • WWC2 has been shown to co-immunoprecipitate with:

    • Gephyrin (inhibitory postsynaptic scaffold)

    • GABAA receptor subunits (γ2 and β2/3)

    • HAP1 and GRIP1 (recycling complex proteins)

  • But not with PSD-95 (excitatory synapse scaffold)

• Proximity ligation assay (PLA):

  • Useful for detecting protein interactions within 40 nm distance

  • Can verify direct interaction between WWC2 and LATS1/2 kinases in the Hippo pathway

• Biochemical fractionation with Western blotting:

  • WWC2 is present in cytosol and plasma membrane/crude synaptosome fractions

  • Combining fractionation with co-IP can reveal compartment-specific interactions

Research has demonstrated that WWC2 enhances phosphorylation of LATS1/2, which in turn phosphorylates YAP1, suppressing its nuclear translocation and transcriptional activity . This mechanistic understanding can inform therapeutic strategies for diseases with dysregulated Hippo signaling.

What are the key considerations when using WWC2 antibodies to study its tumor suppressor role in cancer?

When studying WWC2's tumor suppressor function in cancer research:

This multi-faceted approach can establish WWC2 as both a prognostic marker and potential therapeutic target in HCC.

How can I use WWC2 antibodies to investigate its role in neuronal function and GABAA receptor regulation?

To investigate WWC2's role in neuronal function:

• Synaptic protein interaction studies:

  • WWC2 interacts with the inhibitory postsynaptic scaffold gephyrin but not with PSD-95

  • Use co-IP with WWC2 antibodies to pull down protein complexes from brain tissue or neuronal cultures

  • Probe for GABAA receptor subunits (α2, β2, β3, γ2) and recycling complex proteins (HAP1, GRIP1)

• Subcellular localization in neurons:

  • WWC2 is present in the cytosol and plasma membrane/crude synaptosome fractions but depleted from purified excitatory postsynaptic density (PSD)

  • Use immunofluorescence with WWC2 antibodies to visualize localization at inhibitory synapses

  • Co-stain with gephyrin or GABAA receptor subunits to confirm synaptic localization

• Surface expression analysis:

  • In WWC2 conditional knockout mice, GABAA receptor subunits show increased surface expression

  • Use non-permeabilizing immunofluorescence or surface biotinylation assays with WWC2 and GABAA receptor antibodies

• Functional validation:

  • Combine WWC2 antibody-based biochemical approaches with electrophysiological recordings

  • Loss of WWC2 increases evoked inhibitory synaptic transmission in CA1 pyramidal cells

These approaches have revealed that WWC2 suppresses GABAA receptor incorporation into the plasma membrane and regulates HAP1 and GRIP1, which form a complex promoting GABAA receptor recycling to the membrane .

What controls should I include when using WWC2 antibodies to ensure specificity?

To ensure antibody specificity and reliable interpretation of WWC2 experiments:

• Positive controls:

  • Human brain tissue (high WWC2 expression)

  • Human testis tissue (validated for immunohistochemistry)

  • HEK-293 cells (validated for immunoprecipitation)

• Negative controls:

  • Primary antibody omission

  • Isotype control antibody

  • WWC2 knockdown or knockout samples (if available)

  • Pre-incubation of antibody with immunizing peptide (for blocking controls)

• Isoform considerations:

  • WWC2 exists as seven alternatively spliced isoforms

  • Verify which isoforms your antibody recognizes

  • Multiple bands in Western blot may represent different isoforms rather than non-specific binding

• Cross-reactivity validation:

  • If working with non-human species, verify cross-reactivity

  • WWC2 gene orthologs have been reported in mouse, rat, bovine, frog, chimpanzee, and chicken species

Including these controls helps distinguish specific WWC2 signal from background or cross-reactivity with related proteins like WWC1 (KIBRA), which shares domain structure but has distinct functions .

Why might I observe discrepancies in WWC2 molecular weight across different experiments?

Variability in observed WWC2 molecular weight may result from:

• Protein isoforms:

  • Up to seven isoforms of WWC2 have been reported

  • Canonical WWC2 is 1192 amino acids with a predicted mass of 133.9 kDa

  • Observed molecular weight is approximately 150 kDa in many studies

• Post-translational modifications:

  • Phosphorylation events may alter migration patterns

  • WWC2 is involved in phosphorylation cascades within the Hippo pathway

• Technical factors:

  • Gel percentage (lower percentage gels provide better resolution of high MW proteins)

  • Running buffer composition

  • Voltage and run time

  • Protein marker calibration

• Sample preparation:

  • Denaturation conditions

  • Presence of reducing agents

  • Proteolysis during sample handling

When presenting WWC2 Western blot data, researchers should clearly indicate the observed molecular weight, include appropriate size markers, and discuss potential reasons for discrepancies compared to the predicted molecular weight .

How can WWC2 antibodies be utilized in studying the differential roles of WWC family proteins in synapse-specific regulation?

Recent research has revealed distinct roles for WWC family proteins at different synapse types:

• Comparative analysis approach:

  • WWC2 regulates inhibitory synaptic transmission by suppressing GABAA receptor incorporation into the plasma membrane

  • In contrast, WWC1 (KIBRA) regulates AMPA receptor trafficking at excitatory synapses

  • WWC2 deletion does not affect synaptic AMPAR expression

  • Loss of KIBRA does not affect GABAA receptor membrane expression

• Methodological approach:

  • Use antibodies against both WWC1 and WWC2 in parallel experiments

  • Compare subcellular localization through immunofluorescence

  • Perform co-IP studies to identify synapse-specific binding partners

  • Investigate effects on receptor trafficking through surface biotinylation assays

• Functional validation:

  • Combine biochemical approaches with electrophysiological recordings

  • Use conditional knockout models targeting specific neuronal populations

  • Analyze both spontaneous and evoked synaptic transmission

This research direction is particularly valuable for understanding how related proteins have evolved distinct functions at different synapse types, with implications for targeted therapeutic approaches in neurological disorders .

What are the emerging applications of WWC2 antibodies in understanding its role as a biomarker in cancer progression?

Emerging applications of WWC2 antibodies in cancer research include:

These emerging applications could establish WWC2 not only as a prognostic biomarker but also as a predictive biomarker for targeted therapies in cancers with Hippo pathway dysregulation .