WDFY2 Antibody

What is WDFY2 protein and why is it important in cellular research?

WDFY2 (WD repeat and FYVE domain containing 2) is a 400 amino acid protein with a molecular mass of 45.2 kDa that functions primarily as an adapter protein in endosomal systems. The protein contains WD40 repeats and a FYVE domain that binds specifically to phosphatidylinositol 3-phosphate, allowing it to localize to endosomal membranes . WDFY2 mediates the interaction between the kinase PRKCZ and its substrate VAMP2, enhancing PRKCZ-dependent phosphorylation of VAMP2 .

This protein defines a distinct subset of early endosomes that reside within 100 nm of the plasma membrane, making it a critical marker for studying the earliest stages of endocytic processing . WDFY2 plays a significant role in transferrin uptake, as demonstrated by the impairment of transferrin endocytosis upon WDFY2 silencing . Additionally, recent studies have revealed its importance in metabolic regulation, particularly in insulin sensitivity and glucose metabolism .

What should researchers consider when selecting WDFY2 antibodies?

When selecting WDFY2 antibodies, researchers should consider several critical factors:

• Target epitope and isoform specificity: Determine whether the antibody recognizes the N-terminal, C-terminal, or internal regions of WDFY2. This is particularly important as some commercial antibodies fail to detect shorter WDFY2 isoforms .

• Validated applications: Verify that the antibody has been validated for your specific application (Western blot, immunohistochemistry, immunofluorescence, ELISA) . For example, some antibodies work well for WB but not for IF.

• Species reactivity: Confirm cross-reactivity with your experimental model organism. WDFY2 orthologs have been identified in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

• Purification method: Affinity-purified antibodies typically provide higher specificity. For example, the research described in result #4 employed an affinity-purified rabbit polyclonal antibody raised against a conserved 17-amino acid peptide at the COOH terminus .

• Published validation data: Review literature for successful use of the antibody in similar experimental contexts. The antibody used in result #3 successfully distinguished WDFY2-positive endosomes from EEA1-positive endosomes in deconvolution microscopy .

How can I validate the specificity of my WDFY2 antibody?

Validating antibody specificity is essential for reliable research results. For WDFY2 antibodies, implement the following validation methods:

• Positive and negative controls:

  • Use cell lines with known WDFY2 expression (positive control)

  • Use WDFY2 knockout cells or WDFY2-silenced cells via siRNA (as described in result #3) as negative controls

• Western blot analysis: Verify the antibody detects a band at the expected molecular weight (45.2 kDa for full-length WDFY2) . Be aware that shorter isoforms may also be present.

• Immunodepletion or competition assays: Pre-incubate the antibody with purified WDFY2 protein before immunostaining to confirm signal specificity.

• Orthogonal validation: Compare antibody-based detection with other methods like mass spectrometry or RNA expression data.

• Multiple antibody approach: Use antibodies targeting different epitopes of WDFY2 to confirm consistent localization patterns. Research has shown that commercial antibodies targeting different regions may yield different results, particularly with truncated isoforms .

What are the optimal conditions for using WDFY2 antibodies in Western blot applications?

Based on published research methodologies, the following protocol optimizations are recommended for WDFY2 Western blot applications:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors for protein extraction

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions of WDFY2 with the AKT pathway

• Gel electrophoresis conditions:

  • 10-12% SDS-PAGE gels typically provide optimal resolution for the 45.2 kDa WDFY2 protein

  • If investigating fusion proteins like CDKN2D-WDFY2, use gradient gels (4-15%) to resolve both full-length and truncated forms

• Transfer conditions:

  • Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour in 10% methanol transfer buffer

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary WDFY2 antibody at 1:1000 dilution overnight at 4°C

  • Use species-appropriate HRP-conjugated secondary antibody at 1:5000 dilution

• Detection considerations:

  • Enhanced chemiluminescence (ECL) systems are suitable for standard detection

  • Consider using more sensitive detection systems when examining endogenous WDFY2 in tissues with lower expression levels

Note that researchers studying the CDKN2D-WDFY2 fusion protein encountered difficulties detecting the truncated 7 kDa CDKN2D fragment despite clear transcript evidence, suggesting protein degradation or translational regulation issues .

How can I optimize immunofluorescence protocols for detecting WDFY2 in endosomal compartments?

WDFY2 localizes to a distinct subset of early endosomes near the plasma membrane. For optimal immunofluorescence detection:

• Fixation methods:

  • 4% paraformaldehyde for 15 minutes preserves WDFY2 membrane association

  • Avoid methanol fixation which can disrupt membrane structures

• Permeabilization considerations:

  • Use gentle permeabilization (0.1% Triton X-100 for 5 minutes or 0.1% saponin)

  • Excessive permeabilization may disrupt the delicate endosomal structures

• Colocalization markers:

  • Include markers for proper interpretation:

    • EEA1 (classical early endosome marker that shows minimal overlap with WDFY2-positive vesicles)

    • Fluorescently-labeled transferrin to visualize early endocytic compartments

• Imaging recommendations:

  • Deconvolution microscopy effectively distinguishes WDFY2-positive vesicles from EEA1-positive endosomes

  • TIRF microscopy is particularly valuable for visualizing WDFY2-positive endosomes within 100 nm of the plasma membrane

  • High-resolution confocal microscopy with Airyscan or similar technology for detailed subcellular localization

• Signal amplification:

  • If endogenous detection is challenging, consider tyramide signal amplification (TSA)

  • For dual labeling, ensure primary antibodies are from different species to avoid cross-reactivity

The research in result #3 demonstrated that WDFY2-positive endosomes are more peripherally localized compared to EEA1-positive endosomes, and this distinction is best observed using restored deconvolution microscopy images .

What experimental approaches can determine if WDFY2 antibodies detect fusion proteins like CDKN2D-WDFY2?

The detection of fusion proteins like CDKN2D-WDFY2 requires specialized experimental approaches:

• Multi-antibody strategy:

  • Use antibodies targeting different regions of WDFY2

  • Employ antibodies against both fusion partners (CDKN2D and WDFY2)

  • Compare expression patterns to identify discrepancies that suggest fusion protein presence

• Molecular weight analysis:

  • The CDKN2D-WDFY2 fusion produces a truncated WDFY2 protein of approximately 36 kDa (compared to 45.2 kDa wildtype)

  • Use gradient gels (4-20%) for better resolution of different molecular weight forms

• Epitope-tagged constructs:

  • Create FLAG-tagged or other epitope-tagged versions of the fusion construct

  • Compare detection between epitope antibodies and WDFY2 antibodies as demonstrated in result #2

• Control experiments:

  • Transfect cells with cloned fusion constructs as positive controls

  • Include wildtype WDFY2 expression for comparison

• Mass spectrometry validation:

  • Immunoprecipitate with WDFY2 antibodies and analyze by mass spectrometry to identify truncated peptide sequences

Research has shown that some commercial WDFY2 antibodies failed to detect the short WDFY2 isoform resulting from the CDKN2D-WDFY2 fusion, even though they recognized full-length WDFY2 . This highlights the importance of antibody selection and validation when studying fusion proteins.

How can WDFY2 antibodies be applied in cancer research, particularly in ovarian cancer studies?

WDFY2 antibodies serve as valuable tools in cancer research, especially in studying high-grade serous ovarian cancer where the CDKN2D-WDFY2 fusion gene occurs in approximately 20% of cases :

• Fusion protein detection and classification:

  • Use WDFY2 antibodies in immunohistochemistry to screen patient samples for altered WDFY2 expression patterns

  • Combine with CDKN2D antibodies to identify potential fusion protein expression

  • Develop diagnostic protocols based on altered WDFY2 expression profiles

• Signaling pathway analysis:

  • Employ WDFY2 antibodies in immunoprecipitation to identify altered protein interactions in cancer cells

  • Use phospho-specific antibodies to examine changes in AKT pathway activation, as WDFY2 modulates AKT interactions with substrates

  • Perform reverse phase protein arrays (RPPA) to quantitatively assess changes in signaling networks associated with WDFY2 alterations

• Functional studies:

  • Use WDFY2 antibodies to confirm knockdown efficiency in siRNA experiments investigating functional consequences of WDFY2 loss

  • Monitor changes in protein expression after introducing wildtype or truncated WDFY2 in cancer cells

• Biomarker development:

  • Evaluate WDFY2 antibodies for potential diagnostic applications in identifying patients with CDKN2D-WDFY2 fusion

  • Correlate expression patterns with clinical outcomes and treatment responses

Research has shown that the CDKN2D-WDFY2 fusion alters the PI3K/AKT pathway, which plays a key role in oncogenesis . WDFY2 antibodies can help monitor these changes and potentially identify new therapeutic targets.

What role does WDFY2 play in metabolic research, and how can WDFY2 antibodies contribute to this field?

WDFY2 has emerging significance in metabolic research, particularly in insulin sensitivity and glucose metabolism :

• Insulin signaling studies:

  • Use WDFY2 antibodies to examine protein expression in insulin-responsive tissues (liver, muscle, adipose)

  • Investigate WDFY2 co-localization with insulin receptor and downstream signaling components

• Animal model research:

  • Apply WDFY2 antibodies to validate knockout or knockdown models (as in the systemic Wdfy2 knockout mouse model)

  • Compare WDFY2 expression across metabolic states (fed/fasted, insulin resistant/sensitive)

• Human tissue analysis:

  • Employ WDFY2 antibodies for immunohistochemistry in diabetic vs. non-diabetic tissue samples

  • Correlate WDFY2 expression with metabolic parameters

• Mechanistic investigations:

  • Use WDFY2 antibodies in co-immunoprecipitation studies to identify metabolic interaction partners

  • Examine changes in WDFY2 localization in response to insulin or other metabolic stimuli

• Therapeutic target validation:

  • Monitor WDFY2 expression changes in response to anti-diabetic treatments

  • Screen for compounds that modulate WDFY2 function or expression

Research using a Wdfy2 knockout mouse model has shown WDFY2's importance in insulin sensitivity and glucose metabolism , opening new avenues for diabetes research where WDFY2 antibodies will be essential research tools.

What are common technical challenges when using WDFY2 antibodies and their solutions?

Researchers may encounter several challenges when working with WDFY2 antibodies:

• Isoform detection limitations:

  • Challenge: Commercial antibodies may fail to detect short WDFY2 isoforms

  • Solution: Use antibodies targeting different epitopes or develop custom antibodies against specific regions

• Distinguishing endosomal populations:

  • Challenge: WDFY2-positive endosomes are near but distinct from EEA1-positive endosomes

  • Solution: Use high-resolution imaging techniques like deconvolution microscopy or TIRF microscopy for accurate localization

• Low endogenous expression:

  • Challenge: Endogenous WDFY2 may be expressed at low levels in some tissues

  • Solution: Consider signal amplification methods or increase protein loading for Western blots

• Specificity concerns:

  • Challenge: Cross-reactivity with related WD40/FYVE domain proteins

  • Solution: Include appropriate controls (WDFY2 knockdown/knockout) and verify specificity through multiple detection methods

• Membrane association preservation:

  • Challenge: WDFY2's association with endosomal membranes can be disrupted during sample preparation

  • Solution: Use gentle lysis procedures and avoid strong detergents when membrane association is important

How can I optimize WDFY2 antibody-based experiments for studying endocytosis?

WDFY2 plays a crucial role in early endocytic events, particularly in transferrin uptake . To optimize WDFY2 antibody use in endocytosis research:

• Dual labeling strategies:

  • Combine WDFY2 antibody staining with fluorescent cargo tracking (e.g., fluorescent transferrin)

  • Include markers for different endocytic compartments (clathrin, caveolin, EEA1) to distinguish pathways

• Live cell imaging approaches:

  • Consider using fluorescently-tagged WDFY2 constructs validated against antibody staining patterns

  • Track WDFY2-positive vesicle dynamics in real-time during endocytosis

• Functional assays:

  • Use WDFY2 antibodies to confirm WDFY2 silencing efficiency before performing endocytosis assays

  • Quantify endocytic uptake after WDFY2 manipulation (siRNA, overexpression)

• Subcellular fractionation:

  • Use differential centrifugation to isolate endosomal fractions

  • Verify WDFY2 enrichment in early endosomal fractions using antibodies

• Super-resolution microscopy:

  • Apply techniques like STORM or PALM in combination with WDFY2 antibodies to precisely map WDFY2 distribution on endosomal structures

Research has shown that WDFY2 silencing by siRNA impairs transferrin endocytosis comparably to clathrin silencing, indicating its critical role in early endocytic events .

What controls are essential when using WDFY2 antibodies in research?

Implementing appropriate controls is crucial for generating reliable data with WDFY2 antibodies:

Researchers studying WDFY2's role in endocytosis demonstrated the importance of proper controls by showing that silencing WDFY2 with siRNA (confirmed by Western blot) resulted in reduced transferrin uptake, similar to the effect of clathrin silencing . This functional control validated WDFY2's biological significance in endocytosis.

How can WDFY2 antibodies be used to investigate the PI3K/AKT signaling pathway?

WDFY2 functions as a "docking station" that facilitates interactions between kinases and their substrates, particularly affecting AKT and its substrates . Researchers can leverage WDFY2 antibodies to investigate this pathway:

• Co-immunoprecipitation studies:

  • Use WDFY2 antibodies to pull down protein complexes

  • Analyze precipitated proteins for AKT and its substrates

  • Compare interaction profiles between wildtype and truncated WDFY2

• Signaling cascade analysis:

  • Apply WDFY2 antibodies in combination with phospho-specific antibodies against AKT pathway components

  • Use reverse phase protein arrays (RPPA) to quantitatively assess pathway alterations

  • Compare signaling dynamics in cells expressing wildtype versus short WDFY2 isoforms

• Subcellular localization studies:

  • Investigate co-localization of WDFY2 with activated AKT using immunofluorescence

  • Examine redistribution of AKT pathway components after WDFY2 manipulation

• Functional readouts:

  • Correlate WDFY2 expression/localization with downstream AKT-dependent cellular processes

  • Monitor changes in glucose metabolism, protein synthesis, or cell survival

• Drug response evaluation:

  • Use WDFY2 antibodies to monitor pathway adaptations in response to PI3K/AKT inhibitors

  • Investigate whether WDFY2 status predicts therapeutic response

Research has shown that the short WDFY2 isoform resulting from the CDKN2D-WDFY2 fusion affects the interaction of AKT with its substrates, altering downstream signaling in cancer cells .

What emerging research applications utilize WDFY2 antibodies beyond traditional techniques?

Beyond conventional applications, WDFY2 antibodies are being integrated into emerging technologies:

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions between WDFY2 and binding partners in situ

  • Visualize WDFY2-AKT complexes in single cells with spatial resolution

• CRISPR screening visualization:

  • Use WDFY2 antibodies to visualize phenotypic changes in CRISPR-edited cells

  • Correlate genetic modifications with altered WDFY2 localization or function

• Single-cell proteomics:

  • Apply WDFY2 antibodies in mass cytometry (CyTOF) for high-dimensional analysis of WDFY2 in heterogeneous cell populations

  • Correlate WDFY2 expression with cell state markers

• Tissue spatial transcriptomics integration:

  • Combine WDFY2 antibody staining with spatial transcriptomics to correlate protein localization with gene expression patterns

  • Map WDFY2 distribution across tissue microenvironments

• Organoid and 3D culture systems:

  • Apply WDFY2 antibodies to study endosomal dynamics in physiologically relevant 3D systems

  • Investigate WDFY2 function in polarized cells within organoids

These emerging applications expand the utility of WDFY2 antibodies beyond traditional biochemical and cellular assays, enabling more sophisticated investigations of WDFY2's roles in cellular physiology and disease.