WDFY2 Antibody, HRP conjugated

What is WDFY2 and what cellular functions does it perform?

WDFY2 is an endosomal protein that acts as an adapter mediating interactions between kinase PRKCZ and its substrate VAMP2, increasing PRKCZ-dependent phosphorylation of VAMP2 . It positively regulates adipocyte differentiation by facilitating phosphorylation and inactivation of the anti-adipogenetic transcription factor FOXO1 by kinase AKT1 . WDFY2 plays a critical role in endosomal trafficking, particularly in regulating exocytosis of MT1-MMP (membrane type 1-matrix metalloproteinase) by controlling endosomal sorting of the v-SNARE VAMP3 . It localizes to actin-stabilized endosome tubules positive for the small GTPase RAB4 and shows a preference for highly curved membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P) .

How do HRP-conjugated antibodies function in detection systems?

Horseradish peroxidase (HRP) conjugated antibodies function as signal amplification tools in immunodetection. The HRP enzyme catalyzes the oxidation of substrates in the presence of hydrogen peroxide, resulting in colored precipitates (chromogenic detection), light emission (chemiluminescent detection), or fluorescent signals . When an HRP-conjugated WDFY2 antibody binds to its target protein, the conjugated enzyme allows for visualization through:

• Chemiluminescent detection: Light emission captured by imaging instruments

• Colorimetric detection: Formation of colored precipitates visible without additional equipment

• Fluorescent detection: Generation of fluorescent signals when using tyramide signal amplification systems

The HRP conjugation provides exceptional sensitivity compared to direct detection methods, making it particularly valuable for detecting low-abundance proteins like WDFY2 in subcellular compartments .

What applications are suitable for WDFY2 antibody, HRP conjugated?

WDFY2 antibody, HRP conjugated, can be utilized in several research applications, including:

When selecting an HRP-conjugated WDFY2 antibody, researchers should verify its validated applications, as not all antibodies are suitable for all detection methods .

What cellular localization pattern should I expect when detecting WDFY2?

When using WDFY2 antibodies for cellular localization studies, you should expect the following pattern:

• Primary localization to endosomal structures, particularly subdomains of early endosomes

• Strong signal on endosomal tubules emerging from EEA1-positive endosomes

• Colocalization with RAB4-positive structures (fast-recycling pathway)

• Partial overlap with RAB5-positive regions on endosomes

• Minimal colocalization with RAB11 (slow recycling pathway) and RAB7 (late endosomes)

• Distinct localization from APPL1-positive vesicles near the plasma membrane

• Strong signals at highly curved endosomal membrane regions and tubule bases

Notably, WDFY2 does not completely colocalize with EEA1 but rather localizes to distinct EEA1-negative subdomains on early endosomes .

How does WDFY2's membrane recruitment mechanism affect antibody accessibility?

WDFY2 is recruited to endosomal membranes through its FYVE domain, which specifically binds to PtdIns3P . This membrane interaction affects antibody accessibility in several ways:

• The FYVE domain (amino acids 280-339) may be partially embedded in the membrane when bound to PtdIns3P, potentially masking epitopes in this region

• Antibodies targeting the N-terminal region (amino acids 1-150) may provide better accessibility in fixed cells

• Treatment with PI 3-kinase inhibitors like Wortmannin or SAR405 disrupts WDFY2's endosomal localization, causing it to redistribute to the cytosol

For optimal detection:

• Use antibodies targeting accessible regions (N-terminal or C-terminal domains)

• Ensure proper membrane permeabilization during sample preparation

• Consider fixation methods that preserve membrane architecture while allowing antibody penetration

• Test antibody performance after treatments that disrupt PtdIns3P binding to confirm specificity

Understanding this membrane recruitment mechanism is crucial when designing experiments to study WDFY2's functional domains and protein interactions .

What methodological approaches can distinguish WDFY2's roles in different endosomal subpopulations?

To distinguish WDFY2's roles in different endosomal subpopulations (RAB4-positive vs. RAB5-positive vs. tubular structures), researchers can employ these methodological approaches:

• Super-resolution microscopy techniques:

  • Structured Illumination Microscopy (SIM) to resolve WDFY2's localization to endosomal subdomains

  • Stochastic Optical Reconstruction Microscopy (STORM) to visualize WDFY2 on tubular structures

  • DNA-PAINT to examine colocalization with other endosomal markers

• Multi-color immunofluorescence with domain markers:

  • Co-staining with RAB4/RAB5/RAB11/RAB7 to identify specific endosomal compartments

  • CORONIN1B, WASH, FAM21, VPS26, or VPS35 antibodies to mark actin-stabilized tubular domains

• Live-cell imaging approaches:

  • Stable cell lines expressing GFP-WDFY2 combined with fluorescently tagged RAB proteins

  • Tracking WDFY2-positive tubule dynamics and vesicle formation in real-time

• Functional perturbation experiments:

  • Selective depletion of RAB4 vs. RAB5 to determine WDFY2 dependency

  • Actin cytoskeleton disruption to examine effects on WDFY2-positive tubules

  • PI3K inhibitors to disrupt PtdIns3P binding and assess compartment-specific effects

When using HRP-conjugated antibodies in these approaches, consider secondary detection methods compatible with multicolor analysis or sequential detection protocols.

How can I optimize HRP-conjugated WDFY2 antibody detection in endosomal tubule structures?

Optimizing detection of WDFY2 in endosomal tubule structures requires addressing several technical challenges:

• Fixation optimization:

  • Use paraformaldehyde fixation (4%) to preserve tubular structures

  • Avoid methanol fixation which can disrupt membrane architecture

  • Consider glutaraldehyde addition (0.1-0.5%) for better ultrastructural preservation

• Permeabilization considerations:

  • Gentle detergents (0.1% Triton X-100 or 0.1% saponin) to maintain tubule integrity

  • Shorter permeabilization times to prevent tubule disruption

• Signal amplification strategies:

  • Tyramide signal amplification for enhanced sensitivity in detecting WDFY2 on thin tubular structures

  • SuperBoost tyramide systems for fluorescent visualization after HRP reaction

• Co-staining optimization:

  • Use markers for tubule-stabilizing proteins (CORONIN1B, WASH, FAM21)

  • Include RAB4 staining to identify recycling domains

• Imaging considerations:

  • Z-stack acquisition with optimal step size to capture entire tubular networks

  • Deconvolution processing to improve resolution of thin tubular structures

  • Super-resolution microscopy when available

Research shows that WDFY2 strongly localizes to the base and along the length of endosomal tubules, with particular enrichment at highly curved membrane regions . These structures are relatively long-lived and show accumulations of actin-organizing proteins like CORONIN1B and WASH at their base .

What controls should be included when studying WDFY2's role in MT1-MMP trafficking?

When investigating WDFY2's role in MT1-MMP trafficking using HRP-conjugated antibodies, include these essential controls:

• Antibody specificity controls:

  • WDFY2 knockout or knockdown cells to confirm antibody specificity

  • Peptide competition assays to validate epitope specificity

  • WDFY2 overexpression to verify detection sensitivity

• Functional controls:

  • WDFY2 R315A mutant (disrupts PtdIns3P binding) to confirm loss of endosomal localization

  • VAMP3 knockdown to assess effects on MT1-MMP trafficking independent of WDFY2

  • PI3K inhibitors (Wortmannin or SAR405) to disrupt WDFY2 endosomal recruitment

• Trafficking pathway controls:

  • RAB4 dominant negative constructs to disrupt fast recycling

  • Actin disruption agents to assess effects on WDFY2-positive tubules

  • General endocytosis inhibitors to distinguish recycling from internalization defects

• Readout controls:

  • Surface biotinylation to quantify MT1-MMP plasma membrane levels

  • Gelatin degradation assays to assess functional MT1-MMP activity

  • Comparative analysis with other trafficking cargoes (transferrin receptor)

Research shows that WDFY2 regulates exocytosis of MT1-MMP by controlling endosomal sorting of VAMP3, and loss of WDFY2 leads to enhanced secretion of MT1-MMP and increased cell invasion into extracellular matrix .

What is the recommended protocol for using HRP-conjugated WDFY2 antibodies in western blotting?

For optimal western blot detection of WDFY2 using HRP-conjugated antibodies:

Sample preparation:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Sonicate briefly to shear DNA and reduce sample viscosity

• Centrifuge at 14,000×g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

Gel electrophoresis and transfer:

• Load 20-50 μg of total protein per lane

• Resolve proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membrane (recommended over nitrocellulose for WDFY2)

• Verify transfer efficiency with reversible protein stain

Antibody incubation:

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

• Dilute HRP-conjugated WDFY2 antibody (typically 1:500 to 1:2000, optimize for each antibody)

• Incubate membrane with diluted antibody overnight at 4°C

• Wash extensively with TBST (4 × 10 minutes)

Detection:

• Apply chemiluminescent substrate directly to membrane

• Develop signal using appropriate imaging system

• Expected WDFY2 band size: approximately 45.2 kDa

Critical considerations:

• Include positive control (tissues with known WDFY2 expression)

• Include molecular weight markers to confirm band size

• Validate antibody specificity using WDFY2 knockdown controls

• For weakly expressed WDFY2, consider immunoprecipitation before western blotting

How do I optimize ELISA protocols for WDFY2 detection using HRP-conjugated antibodies?

For optimizing ELISA protocols with HRP-conjugated WDFY2 antibodies:

Direct ELISA protocol:

• Coat high-binding 96-well plate with sample diluted in carbonate buffer (pH 9.6) overnight at 4°C

• Wash 3× with PBST (PBS + 0.05% Tween-20)

• Block with 1-5% BSA in PBS for 1-2 hours at room temperature

• Wash 3× with PBST

• Add HRP-conjugated WDFY2 antibody diluted in blocking buffer (start with manufacturer's recommended dilution)

• Incubate 1-2 hours at room temperature or overnight at 4°C

• Wash 5× with PBST

• Add TMB substrate and monitor color development

• Stop reaction with 2N H₂SO₄ and read absorbance at 450 nm

Sandwich ELISA optimization:

• Coat plate with capture antibody (non-conjugated WDFY2 antibody) overnight

• Block as above

• Add samples containing WDFY2 protein

• Add HRP-conjugated WDFY2 antibody recognizing a different epitope

• Develop and read as above

Optimization parameters:

• Antibody concentration: Titrate from 1:200 to 1:5000

• Incubation temperature: Compare room temperature vs. 4°C

• Incubation time: Test 1, 2, and 4 hours vs. overnight

• Blocking agent: Compare BSA, milk, and commercial blockers

• Substrate: Compare TMB, ABTS, or OPD for optimal signal-to-noise ratio

Critical considerations:

• Use antibodies recognizing different epitopes for sandwich ELISA

• Include standard curve with recombinant WDFY2 protein

• Run parallel negative controls omitting primary antibody

• Test sensitivity by serial dilution of positive control

What approaches should I use to troubleshoot weak or non-specific signals with HRP-conjugated WDFY2 antibodies?

When encountering weak or non-specific signals with HRP-conjugated WDFY2 antibodies, implement this systematic troubleshooting approach:

For weak signals:

• Antibody concentration:

  • Increase antibody concentration (reduce dilution factor)

  • Extended incubation time (overnight at 4°C)

• Signal amplification:

  • Try more sensitive substrate (SuperSignal West Femto vs. Pico)

  • Consider tyramide signal amplification systems for immunostaining

  • For western blots, increase protein loading (50-100 μg)

• Sample preparation:

  • Optimize extraction buffer (RIPA vs. NP-40 vs. specialized buffers)

  • Add protease inhibitors to prevent degradation

  • Consider subcellular fractionation to enrich endosomal compartments

• Detection conditions:

  • Extend substrate incubation time

  • Optimize exposure time for western blots

  • Try alternative HRP substrates with higher sensitivity

For non-specific signals:

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.5% Tween-20 to antibody diluent

• Antibody specificity:

  • Validate with WDFY2 knockout/knockdown controls

  • Perform peptide competition assays

  • Try antibodies targeting different WDFY2 epitopes

• Washing conditions:

  • Increase number and duration of washes

  • Use higher Tween-20 concentration in wash buffer (0.1%)

  • Include salt wash steps (high stringency washes)

• Cross-reactivity reduction:

  • Pre-absorb antibody with cell/tissue lysates from WDFY2-negative samples

  • Use highly purified antibodies when available

  • Test antibodies validated for specific applications

Remember that WDFY2 is expressed at modest levels in many cell types, making detection challenging. Use positive controls from tissues known to express WDFY2 and consider enrichment strategies for low-abundance samples.

How do I quantify and interpret changes in WDFY2 localization using immunofluorescence?

To quantify and interpret WDFY2 localization changes in immunofluorescence experiments:

Quantification methods:

• Colocalization analysis:

  • Calculate Pearson's correlation coefficient between WDFY2 and endosomal markers

  • Determine Manders' overlap coefficient to assess proportion of WDFY2 overlapping with RAB4/RAB5/EEA1

  • Use object-based colocalization for discrete structures

• Morphological analysis:

  • Measure number, length, and branching of WDFY2-positive tubules

  • Quantify ratio of tubular versus vesicular WDFY2 localization

  • Calculate WDFY2 enrichment at tubule bases versus limiting membrane

• Distribution analysis:

  • Measure WDFY2 intensity gradients from plasma membrane to perinuclear regions

  • Quantify distance of WDFY2-positive endosomes from cell edge

  • Analyze spatial relationship with actin cytoskeleton

Data analysis workflow:

• Collect z-stack images under identical acquisition settings

• Apply appropriate background subtraction

• Define regions of interest (entire cell, peripheral region, perinuclear region)

• Threshold WDFY2 signal and endosomal markers consistently across samples

• Apply colocalization or morphological analysis algorithms

• Export numerical data for statistical analysis

• Compare experimental conditions using appropriate statistical tests

Interpretation guidelines:

• WDFY2 normally localizes to subdomains on EEA1-positive endosomes and endosomal tubules

• Increased cytosolic WDFY2 suggests disrupted PtdIns3P binding or endosomal recruitment

• Enhanced tubular localization may indicate altered endosomal recycling

• Loss of colocalization with RAB4 suggests defects in fast recycling pathways

• Changes in WDFY2-VAMP3 association may affect MT1-MMP trafficking and matrix degradation

WDFY2 localization changes should be interpreted in context of functional outcomes, such as alterations in cargo trafficking, cell migration, or matrix degradation.

What considerations are important when analyzing WDFY2 function in different cell types?

When analyzing WDFY2 function across different cell types, consider these important factors:

Expression level variations:

• WDFY2 expression levels vary significantly between cell types

• Western blot quantification should be normalized to housekeeping proteins

• Use absolute quantification methods (recombinant protein standards) for direct comparisons

Functional context differences:

Technical considerations:

• Optimize antibody dilutions for each cell type

• Adjust fixation protocols based on cell type (epithelial vs. fibroblast vs. adipocyte)

• Consider endogenous expression levels when interpreting knockdown/overexpression phenotypes

• Use cell type-specific markers to contextualize WDFY2 localization patterns

Biological context assessment:

• Evaluate relevant trafficking pathways (VAMP3, MT1-MMP) in context of cell-specific functions

• Consider interaction with tissue-specific matrix components

• Assess cell type-specific responses to WDFY2 manipulation

• Examine relevant signaling cascades (AKT, insulin signaling) in appropriate cell types

When comparing WDFY2 functions across cell types, use consistent experimental approaches and quantification methods to enable valid comparisons of phenotypes and molecular mechanisms.

How do I integrate WDFY2 localization data with functional outcomes in invasion and trafficking studies?

To meaningfully integrate WDFY2 localization data with functional outcomes in invasion and trafficking studies, implement this comprehensive approach:

Correlation analysis framework:

• Matched sample analysis:

  • Perform parallel assays on the same cell populations

  • Correlate WDFY2 subcellular distribution with quantitative functional readouts

  • Use regression analysis to identify predictive localization patterns

• Temporal relationship studies:

  • Track WDFY2 localization changes over time

  • Monitor corresponding changes in MT1-MMP surface levels

  • Measure subsequent matrix degradation or invasion events

  • Establish cause-effect relationships through time-course experiments

• Structure-function analysis:

  • Compare wild-type WDFY2 versus functional mutants (R315A)

  • Correlate specific domain requirements with functional outcomes

  • Map critical interaction sites for VAMP3 binding and functional regulation

Integrated experimental approach:

For invasion studies:

• Quantify WDFY2 localization to RAB4-positive endosomes and tubules

• Measure MT1-MMP surface levels by biotinylation or flow cytometry

• Assess matrix degradation using fluorescent gelatin degradation assays

• Quantify 3D invasion through matrigel or collagen matrices

• Calculate correlation coefficients between WDFY2 localization metrics and functional outcomes

For trafficking studies:

• Track WDFY2-positive endosomal tubule dynamics

• Monitor VAMP3 trafficking in same cells

• Measure cargo recycling rates (MT1-MMP or transferrin)

• Correlate tubule formation frequency with recycling efficiency

• Use live-cell imaging to establish temporal relationships

Data integration schema:

Research shows that loss of WDFY2 leads to enhanced secretion of MT1-MMP and allows cells to actively invade into ECM, connecting WDFY2's endosomal functions directly to invasive potential .

What emerging technologies could enhance the study of WDFY2 using HRP-conjugated antibodies?

Several emerging technologies could significantly enhance WDFY2 studies using HRP-conjugated antibodies:

• Proximity labeling approaches:

  • HRP-APEX technology for spatially restricted protein labeling

  • Combining HRP-WDFY2 antibodies with tyramide-biotin for selective labeling of proximal proteins

  • Identification of transient WDFY2 interaction partners on endosomal tubules

• Correlative light and electron microscopy (CLEM):

  • Using HRP-conjugated antibodies for both fluorescence and EM contrast

  • Diaminobenzidine (DAB) polymerization for ultrastructural visualization

  • Nanoscale resolution of WDFY2 on endosomal tubule membranes

• Super-resolution microscopy enhancements:

  • Combination of HRP-mediated signal amplification with STORM/PALM

  • Expansion microscopy for physical magnification of subcellular structures

  • Multi-color DNA-PAINT for simultaneous visualization of multiple endosomal markers

• Live-cell applications:

  • Split-HRP complementation assays for studying WDFY2-VAMP3 interactions

  • HRP-mediated activation of fluorogenic probes for dynamic studies

  • Intrabody approaches using HRP-tagged nanobodies against WDFY2

• Quantitative approaches:

  • Multiplexed ion beam imaging (MIBI) using metal-conjugated WDFY2 antibodies

  • Mass cytometry for single-cell analysis of WDFY2 in heterogeneous populations

  • Highly multiplexed immunofluorescence using sequential HRP-antibody labeling

These technologies could provide unprecedented insights into WDFY2's dynamic localization and function at endosomal tubules, particularly in relation to its role in controlling MT1-MMP trafficking and cell invasion .

How might our understanding of WDFY2's role in disease progression influence antibody-based detection strategies?

Emerging understanding of WDFY2's role in disease progression could significantly influence antibody-based detection strategies:

Cancer-focused applications:

• Given WDFY2's role in regulating MT1-MMP secretion and cell invasion , antibodies targeting specific functional domains could serve as diagnostic tools for invasive tumors

• Development of phospho-specific antibodies to detect activated WDFY2 in tumor samples

• Creation of antibody panels to simultaneously assess WDFY2 and its key partners (VAMP3, MT1-MMP) in tumor biopsies

Metabolic disease applications:

• Due to WDFY2's role in adipocyte differentiation and insulin signaling , specialized antibodies to detect tissue-specific isoforms or modifications

• Multiplex detection systems to simultaneously assess WDFY2, AKT phosphorylation, and FOXO1 localization

• Development of antibodies specifically detecting WDFY2 in insulin-responsive tissues

Technical innovations:

• Domain-specific antibodies to distinguish active vs. inactive WDFY2 conformations

• Engineered antibodies with enhanced penetration into endosomal compartments

• Bifunctional antibodies targeting WDFY2 and trafficking cargo simultaneously

• Spatially-resolved antibody detection using spatial transcriptomics platforms

Clinical translation considerations:

• Standardization of WDFY2 detection for potential biomarker applications

• Validation across diverse tissue types and disease states

• Correlation of WDFY2 alterations with disease progression and therapeutic responses

As research continues to uncover WDFY2's roles in cancer invasion, metabolic regulation, and potentially neurodegenerative diseases (given its endosomal functions), antibody technologies will need to evolve to address specific disease-relevant modifications, interactions, and subcellular localizations.