WDPCP Antibody, Biotin conjugated

What is WDPCP and why is it significant in cellular research?

WDPCP (WD repeat-containing and planar cell polarity effector protein fritz homolog) is a protein that plays a crucial role in planar cell polarity signaling and is alternatively known as Bardet-Biedl syndrome 15 protein (BBS15), C2orf86, or FRITZ . The significance of WDPCP in cellular research stems from its essential functions in regulating cell polarity, which is fundamental for proper tissue development and organization.

WDPCP mediates epicardial epithelial-mesenchymal transition (EMT) and cardiac cell migration during coronary artery remodeling . Its disruption can lead to cardiomyocyte disorganization in congenital heart defects (CHDs) . The protein serves as an upstream regulator of the MAPK/ERK signaling pathway, which is critical for endothelial function and coronary artery development . This regulatory role positions WDPCP as a significant target for understanding developmental disorders and pathological conditions involving tissue architecture and cellular organization.

What are the advantages of using biotin-conjugated antibodies in WDPCP research?

Biotin-conjugated antibodies offer several methodological advantages in WDPCP research:

• Enhanced detection sensitivity: The high-affinity interaction between biotin and streptavidin (Kd ≈ 10^-15 M) provides exceptional signal amplification, allowing detection of low-abundance WDPCP protein in complex biological samples .

• Versatile detection systems: Biotin-conjugated WDPCP antibodies can be detected using various streptavidin-conjugated reporter molecules (fluorophores, enzymes, quantum dots), providing flexibility across multiple experimental platforms .

• Stability and compatibility: The biotin modification maintains antibody stability while offering compatibility with various buffer systems and experimental conditions .

• Immobilization capabilities: Biotin-conjugated WDPCP antibodies can be efficiently immobilized on streptavidin-coated surfaces for applications such as protein capture, pull-down assays, and immunoprecipitation studies .

• Multiplex potential: The biotin-streptavidin system enables multiplex detection strategies when investigating WDPCP alongside other proteins in signaling pathways .

How does WDPCP function in cell signaling pathways?

WDPCP functions as a critical component in multiple cell signaling networks:

• MAPK/ERK pathway regulation: WDPCP serves as an upstream regulator of MAPK/ERK signaling, with overexpression of WDPCP enhancing the levels of phospho-p38 and phospho-ERK . This regulatory relationship is unidirectional, as MAPK activation does not affect WDPCP expression levels.

• EMT modulation: WDPCP positively regulates the epithelial-mesenchymal transition process in human cardiac microvascular endothelial cells (HCMECs). Overexpression of WDPCP restores expression of mesenchymal proteins like vimentin and N-cadherin while suppressing epithelial markers such as E-cadherin .

• Cell migration control: WDPCP promotes cell migration and invasion capabilities, particularly in endothelial cells. High leucine levels have been shown to impair mobility and EMT in HCMECs by repressing WDPCP expression .

• Planar cell polarity signaling: As implied by its name, WDPCP plays an instrumental role in planar cell polarity pathways that govern cell organization within tissue planes, which is essential for proper angiogenic organization of endothelial cells, arterial remodeling, and heart morphogenesis .

• Epicardial adipose tissue regulation: WDPCP overexpression has been demonstrated to reduce epicardial adipose tissue volume, suggesting a role in regulating adipocyte differentiation or accumulation .

What are the optimal storage and handling conditions for WDPCP antibody, biotin conjugated?

The optimal storage and handling conditions for WDPCP antibody, biotin conjugated are critical for maintaining its functionality and specificity:

Storage Requirements:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as they can compromise antibody integrity

• For short-term storage (less than 1 month), 4°C is acceptable but not recommended for longer periods

Buffer Composition:
The antibody is typically supplied in a stabilizing buffer containing:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 as a preservative

Handling Recommendations:

• When removing from frozen storage, thaw the antibody slowly on ice

• Centrifuge briefly to collect solution at the bottom of the tube before opening

• Aliquot into smaller volumes for multiple use to minimize freeze-thaw cycles

• Maintain sterile conditions when handling to prevent microbial contamination

• Return to appropriate storage temperature immediately after use

Adhering to these storage and handling guidelines ensures optimal antibody performance and extends its useful shelf life for experimental applications.

What is the recommended protocol for using WDPCP antibody, biotin conjugated in Western blot analysis?

Recommended Western Blot Protocol for WDPCP Antibody, Biotin Conjugated:

Sample Preparation:

• Extract proteins using RIPA buffer containing protease inhibitor cocktail

• Quantify protein concentration using BCA assay

• Denature 5μg of protein sample for SDS-PAGE analysis

Gel Electrophoresis and Transfer:

• Resolve proteins on a 12% polyacrylamide gel

• Transfer separated bands to a PVDF membrane

Blocking and Antibody Incubation:

• Block membrane with 5% skimmed milk for 1 hour at room temperature

• Incubate with anti-WDPCP biotin-conjugated antibody (1:1000 dilution) at 4°C for 24 hours

• Wash membrane 3x with TBST (10 minutes each)

• Incubate with streptavidin-HRP (1:5000) for 1 hour at room temperature

• Wash membrane 3x with TBST (10 minutes each)

Detection and Analysis:

• Develop using Western blot chemiluminescence kit

• Image bands using a GelDoc imaging system

• Quantify band intensities using ImageJ software

• Normalize WDPCP band intensity to β-Actin (loading control)

Key Considerations:

• Include positive and negative controls

• Optimize antibody concentration if signal-to-noise ratio is suboptimal

• For multiplex detection, strip and reprobe the membrane or use spectrally distinct detection systems

• For phosphorylation studies, consider using phospho-specific antibodies alongside the WDPCP antibody

This protocol has been validated for detecting WDPCP protein in both cell lysates and plasma samples in research investigating cell polarity and MAPK signaling pathways .

How can researchers optimize ELISA protocols using WDPCP antibody, biotin conjugated?

Optimized ELISA Protocol for WDPCP Antibody, Biotin Conjugated:

Plate Preparation:

• Coat 96-well high-binding ELISA plate with capture antibody against WDPCP (1-2 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C

• Wash 3x with PBS containing 0.05% Tween-20 (PBST)

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

Sample Processing:

• Prepare samples (cell lysates or biological fluids) in appropriate dilution buffer

• Create standard curve using recombinant WDPCP protein (range: 0-1000 ng/ml)

• Add 100 μl of samples and standards to respective wells

• Incubate for 2 hours at room temperature with gentle shaking

Detection System:

• Wash 5x with PBST

• Add biotin-conjugated WDPCP antibody (1:5000 dilution) in 1% BSA/PBS

• Incubate for 1 hour at room temperature

• Wash 5x with PBST

• Add streptavidin-HRP (1:10,000) in 1% BSA/PBS

• Incubate for 30 minutes at room temperature

• Wash 5x with PBST

Signal Development and Analysis:

• Add 100 μl TMB substrate solution

• Allow color development (typically 5-15 minutes) in the dark

• Stop reaction with 100 μl of 2N H₂SO₄

• Measure absorbance at 450 nm with 570 nm as reference wavelength

Optimization Strategies:

• Antibody titration: Test different concentrations of the biotin-conjugated WDPCP antibody (1:1000 to 1:10,000) to determine optimal signal-to-noise ratio

• Sample dilution series: Run multiple dilutions of test samples to ensure readings fall within the linear range of the standard curve

• Incubation time adjustment: Optimize incubation times for sample and antibody steps to maximize specific signal while minimizing background

• Buffer optimization: Test different blocking agents (BSA, milk, commercial blockers) to reduce non-specific binding

• Temperature control: Compare room temperature vs. 37°C incubation for critical steps to enhance sensitivity

This protocol has been validated for detecting WDPCP in research applications studying cellular signaling pathways and is compatible with the biotin-conjugated format of the antibody .

How can WDPCP antibody, biotin conjugated be used to investigate WDPCP-MAPK signaling interactions?

WDPCP antibody, biotin conjugated offers several methodological approaches to investigate the WDPCP-MAPK signaling interactions:

Co-Immunoprecipitation Studies:

• Use biotin-conjugated WDPCP antibody to precipitate WDPCP protein complexes from cell lysates using streptavidin-coated magnetic beads

• Perform Western blot analysis on the precipitated complexes to detect associated MAPK pathway components (ERK1/2, p38)

• Compare protein interactions under various stimulation conditions to map signaling dynamics

Signaling Pathway Analysis:

• Treat cells with MAPK pathway modulators (activators like C16-PAF or inhibitors)

• Detect changes in WDPCP expression and phosphorylation status using the biotin-conjugated antibody

• Simultaneously monitor phospho-p38 and phospho-ERK levels to establish correlation with WDPCP levels

Kinase-Catalyzed Biotinylation Approach:

• Implement kinase-catalyzed biotinylation with ATP-biotin to map phosphorylation events in the WDPCP-MAPK pathway

• Enrich biotinylated proteins using streptavidin resin

• Detect WDPCP and MAPK components (ERK1/2, AKT1) in eluates using specific antibodies

• Quantify changes in phosphorylation patterns under different stimulation conditions

Research findings from these approaches have revealed:

• WDPCP acts as an upstream regulator of MAPK/ERK signaling, as WDPCP overexpression promotes phospho-p38 and phospho-ERK levels

• MAPK activator treatment does not affect WDPCP expression, confirming the unidirectional relationship

• Both WDPCP overexpression and MAPK activation reduce epicardial adipose tissue volume (EATV) in high-leucine diet models

• WDPCP and MAPK signaling both mediate suppressive effects on cell migration and EMT in cardiac microvascular endothelial cells

This methodological framework enables researchers to dissect the complex regulatory relationships between WDPCP and MAPK signaling components, providing insights into their roles in cardiac development and pathological conditions.

What are the validated applications of WDPCP antibody, biotin conjugated in cardiac development research?

WDPCP antibody, biotin conjugated has been validated for several specific applications in cardiac development research:

Congenital Heart Defect (CHD) Studies:

• Protein Expression Analysis: Western blot detection of WDPCP levels in plasma samples from CHD patients compared to healthy controls, revealing significant downregulation in disease states

• Epicardial Adipose Tissue Volume (EATV) Assessment: Correlation of WDPCP expression with EATV measurements in cardiac imaging studies, showing inverse relationships

• Model Systems: Detection of WDPCP in neonatal mice from high-leucine diet groups that develop CHD-like features

Epithelial-Mesenchymal Transition (EMT) Analysis:

• Protein Marker Profiles: Monitoring changes in epithelial markers (E-cadherin) and mesenchymal markers (vimentin, N-cadherin) in relation to WDPCP expression

• Rescue Experiments: Detection of WDPCP levels following overexpression interventions in high-leucine models to restore normal cardiac development

Endothelial Cell Function Studies:

• Human Cardiac Microvascular Endothelial Cells (HCMECs): Assessment of WDPCP expression and its impact on endothelial cell polarity, migration, and invasion

• EMCN Regulation: Investigation of WDPCP's role in regulating EMCN (endomucin) levels in endothelial cells under various conditions

MAPK Pathway Interactions:

• Signaling Cascade Analysis: Evaluation of WDPCP's position in the MAPK signaling hierarchy through phosphorylation status assessment of downstream targets

• Intervention Studies: Measuring changes in WDPCP expression and function following treatment with MAPK activators (C16-PAF)

Key Research Findings:

• WDPCP expression is reduced in CHD patients and in high-leucine-induced CHD models

• WDPCP overexpression ameliorates high-leucine-induced cardiac defects

• WDPCP functions upstream of MAPK/ERK signaling in cardiac development

• WDPCP positively regulates endothelial cell migration and EMT processes

• Disruption of WDPCP expression impairs both endothelial angiogenesis and cardiac septation

These validated applications demonstrate the utility of biotin-conjugated WDPCP antibody in elucidating the molecular mechanisms underlying cardiac development and congenital heart defects.

How can researchers utilize WDPCP antibody, biotin conjugated in cell migration and EMT studies?

Researchers can utilize WDPCP antibody, biotin conjugated in cell migration and EMT studies through several methodological approaches:

Cell Migration Assays:

• Wound Healing Analysis:

  • Create a scratch in confluent cell monolayers

  • Treat cells with factors affecting WDPCP expression

  • Monitor wound closure over time

  • Use the biotin-conjugated WDPCP antibody in parallel immunofluorescence studies to correlate migration capacity with WDPCP levels

• Transwell Migration Assay:

  • Employ Boyden chamber/transwell inserts to assess directional cell migration

  • Manipulate WDPCP expression through overexpression or knockdown

  • Detect WDPCP protein levels in migrated vs. non-migrated cells using the biotin-conjugated antibody and streptavidin-fluorophore detection

EMT Marker Profiling:

• Western Blot Analysis:

  • Simultaneously detect WDPCP and EMT markers (E-cadherin, N-cadherin, vimentin)

  • Normalize protein expression to β-actin

  • Quantify relative expression levels using densitometry

  • Compare expression patterns across experimental conditions

• Immunofluorescence Co-localization:

  • Perform multiplex immunofluorescence using biotin-conjugated WDPCP antibody with streptavidin-fluorophore

  • Co-stain for EMT markers using differently labeled secondary antibodies

  • Analyze subcellular localization and expression patterns

  • Quantify co-localization coefficients to establish relationship between WDPCP and EMT markers

WDPCP Manipulation Studies:

• Overexpression Experiments:

  • Transfect cells with WDPCP expression vectors

  • Verify overexpression using the biotin-conjugated antibody

  • Assess changes in cell migration and EMT marker expression

  • Correlation analysis between WDPCP levels and migration/EMT parameters

• RNAi Knockdown:

  • Deplete WDPCP using siRNA or shRNA approaches

  • Confirm knockdown efficiency using the biotin-conjugated antibody

  • Evaluate effects on migration capacity and EMT marker expression

  • Rescue experiments to establish specificity of observed phenotypes

Research Findings Table:

This methodological framework allows researchers to comprehensively investigate WDPCP's role in regulating cell migration and EMT processes, particularly in the context of cardiac development and congenital heart defects .

How can kinase-catalyzed biotinylation be combined with WDPCP antibody studies for phosphorylation dynamics analysis?

Kinase-catalyzed biotinylation provides a powerful approach to study WDPCP phosphorylation dynamics when strategically combined with WDPCP antibody studies:

Methodological Integration:

• ATP-Biotin Labeling of Dynamic Phosphosites:

  • Incubate cell lysates with ATP-biotin (2 mM) to enable kinase-catalyzed transfer of biotin to dynamically changing phosphosites

  • This approach specifically labels sites undergoing active phosphorylation/dephosphorylation

  • Include appropriate controls: unstimulated lysates, inhibitor-treated lysates, and no-ATP-biotin controls

• Enrichment of Biotinylated Proteins:

  • Dilute reaction mixtures with phosphate binding buffer

  • Incubate with streptavidin resin to capture biotinylated proteins

  • Collect flow-through for comparison

  • Elute bound proteins under denaturing conditions

• Dual Detection Strategy:

  • Perform parallel immunoblotting using:
    a) Anti-WDPCP antibody to detect total WDPCP protein
    b) Streptavidin-HRP to detect biotinylated phosphosites
    c) Phospho-specific antibodies for MAPK pathway components

• Quantitative Analysis:

  • Normalize signal intensities to input samples

  • Calculate relative phosphorylation levels across experimental conditions

  • Apply statistical analysis using unpaired t-tests to determine significance

Experimental Design for WDPCP Phosphorylation Studies:

Advanced Applications:

• Temporal Dynamics Assessment:

  • Stimulate cells for varying time periods (5, 15, 30, 60 minutes)

  • Apply kinase-catalyzed biotinylation at each timepoint

  • Monitor changes in WDPCP phosphorylation patterns over time

  • Correlate with activation states of MAPK pathway components

• Pathway Cross-talk Analysis:

  • Selectively inhibit specific branches of signaling networks

  • Assess impact on WDPCP phosphorylation via kinase-catalyzed biotinylation

  • Map interdependencies between WDPCP and other signaling components

• Phosphosite Identification:

  • Combine with LC-MS/MS analysis of biotinylated peptides

  • Identify specific WDPCP residues undergoing phosphorylation

  • Correlate site-specific modifications with functional outcomes

This integrated approach provides researchers with unprecedented resolution of WDPCP phosphorylation dynamics, allowing correlation between phosphorylation status and functional outcomes in cell migration, EMT, and cardiac development contexts.

What are the considerations for multiplex analysis involving WDPCP antibody, biotin conjugated?

Multiplex analysis involving WDPCP antibody, biotin conjugated requires careful consideration of several technical and experimental factors:

Panel Design Considerations:

• Spectral Compatibility:

  • When choosing additional detection reagents, ensure spectral separation between fluorophores

  • Consider the excitation/emission spectra of streptavidin conjugates (typically used with biotin-conjugated antibodies)

  • Select complementary fluorophores for co-detection of other targets

• Antibody Cross-Reactivity:

  • Validate antibody specificity individually before multiplexing

  • Test for potential cross-reactivity between antibodies in the panel

  • Consider using antibodies raised in different host species to facilitate detection

• Signal Intensity Balancing:

  • Adjust antibody concentrations to achieve comparable signal intensities

  • Account for differences in target abundance (WDPCP vs. MAPK components)

  • Optimize exposure/gain settings for each detection channel

Multiplex Experimental Approaches:

• Immunofluorescence Multiplexing:

  • Use streptavidin-fluorophore conjugate to detect biotin-WDPCP antibody

  • Employ directly labeled antibodies for other targets

  • Apply sequential staining for targets requiring same-species antibodies

  • Include appropriate controls for autofluorescence and spectral overlap

• Flow Cytometry Applications:

  • Utilize streptavidin-fluorophore for WDPCP detection

  • Combine with directly conjugated antibodies against MAPK components

  • Perform proper compensation controls for each fluorophore

  • Include FMO (fluorescence minus one) controls for accurate gating

• Multiplex Western Blotting:

  • Implement sequential probing with stripping between antibodies

  • Consider fluorescent Western blotting with spectrally distinct detection systems

  • Validate that stripping does not affect subsequent antibody binding

Technical Optimization Table:

Validated Multiplex Applications:

• WDPCP-MAPK Pathway Analysis:

  • Simultaneous detection of WDPCP, phospho-p38, and phospho-ERK

  • Correlation of expression patterns in response to stimuli or inhibitors

  • Subcellular co-localization assessment

• EMT Marker Profiling:

  • Co-detection of WDPCP with epithelial markers (E-cadherin) and mesenchymal markers (N-cadherin, vimentin)

  • Quantitative analysis of marker expression patterns

  • Correlation with cell migration phenotypes

By addressing these considerations, researchers can develop robust multiplex analysis protocols involving biotin-conjugated WDPCP antibody, enabling comprehensive investigation of complex signaling networks and cellular processes.

How can researchers validate the specificity and sensitivity of WDPCP antibody, biotin conjugated in their experimental systems?

Researchers can implement a comprehensive validation strategy to ensure the specificity and sensitivity of WDPCP antibody, biotin conjugated in their experimental systems:

Specificity Validation Approaches:

• Knockout/Knockdown Controls:

  • Compare antibody staining in wildtype vs. WDPCP-knockout or WDPCP-knockdown samples

  • Verify significant reduction or elimination of signal in the absence of target protein

  • Include partial knockdown samples to assess signal proportionality to protein levels

• Overexpression Validation:

  • Test antibody in cells transfected with WDPCP expression vector vs. empty vector controls

  • Confirm increased signal intensity corresponding to overexpression levels

  • Verify correct molecular weight of detected band by Western blot

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (WDPCP 621-737AA)

  • Apply pre-absorbed antibody in parallel with regular antibody

  • Observe reduced or eliminated signal with pre-absorbed antibody

• Multiple Antibody Concordance:

  • Compare detection patterns with alternative WDPCP antibodies targeting different epitopes

  • Consistent patterns across different antibodies increase confidence in specificity

Sensitivity Assessment Methods:

• Dilution Series Analysis:

  • Prepare serial dilutions of samples containing WDPCP protein

  • Determine limit of detection (LOD) and limit of quantification (LOQ)

  • Generate standard curve with recombinant WDPCP protein

• Signal-to-Noise Optimization:

  • Test various blocking agents (BSA, milk, commercial blockers)

  • Optimize antibody concentration through titration experiments

  • Evaluate different detection systems (chemiluminescence, fluorescence)

• Cross-Reactivity Testing:

  • Assess antibody performance in samples from different species (though this particular antibody is specific for human WDPCP)

  • Test in different cell/tissue types with varying WDPCP expression levels

  • Evaluate potential cross-reactivity with related proteins

Validation Data Analysis and Documentation:

Experimental System-Specific Validation:

• Cell Line Validation:

  • Test antibody in cell lines known to express WDPCP (e.g., HCMECs)

  • Include negative control cell lines with minimal WDPCP expression

  • Verify detection of WDPCP induction upon relevant stimulation

• Tissue Sample Validation:

  • Test in tissue sections with known WDPCP expression patterns

  • Include appropriate negative controls (e.g., isotype control)

  • Verify expected subcellular localization

• Functional Validation:

  • Confirm that detected WDPCP correlates with expected biological functions

  • Verify relationships with known interaction partners (MAPK components)

  • Ensure detected changes correspond to functional outcomes in cell migration and EMT

Through this comprehensive validation strategy, researchers can confidently apply the biotin-conjugated WDPCP antibody in their experimental systems, ensuring reliable and reproducible results in their investigations of WDPCP biology and function.

What are common challenges in WDPCP antibody experiments and their solutions?

Common Challenges and Solutions in WDPCP Antibody Experiments:

Experimental Troubleshooting Strategies:

• Western Blot Optimization:

  • Adjust protein loading (5-20 μg range)

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Optimize transfer conditions (time, voltage, buffer composition)

  • Try gradient gels for better resolution of WDPCP

• ELISA Troubleshooting:

  • Implement plate-washing optimization (number of washes, wash volume)

  • Test different plate types (high-binding vs. medium-binding)

  • Adjust incubation temperatures (4°C, RT, 37°C)

  • Optimize detection system (substrate development time)

• Immunofluorescence Challenges:

  • Test different fixation methods (paraformaldehyde, methanol)

  • Optimize permeabilization conditions

  • Implement antigen retrieval if necessary

  • Adjust mounting media to reduce photobleaching

• Co-IP Specific Issues:

  • Test different lysis buffers to preserve protein interactions

  • Optimize bead type and volume

  • Adjust binding and washing stringency

  • Consider crosslinking to stabilize transient interactions

Special Considerations for WDPCP Studies:

• WDPCP exhibits relatively low expression in some cell types; consider enrichment strategies before detection

• As a regulatory protein, WDPCP levels may fluctuate with cell cycle or differentiation status

• Post-translational modifications may affect antibody recognition; consider phosphatase treatment controls

• The biotin-streptavidin interaction can be disrupted by high salt or detergent concentrations; optimize buffer conditions accordingly

Implementing these troubleshooting strategies will help researchers overcome common challenges in WDPCP antibody experiments and obtain reliable, reproducible results.

How can researchers differentiate between WDPCP splice variants or post-translationally modified forms?

Methodological Approaches to Differentiate WDPCP Variants:

• Gel Resolution Strategies:

  • Employ gradient gels (4-15% or 4-20%) to maximize separation of different WDPCP forms

  • Use large-format gels for enhanced resolution of closely migrating variants

  • Implement Phos-tag™ acrylamide gels to separate phosphorylated from non-phosphorylated forms

  • Run samples on lower percentage gels (6-8%) with extended separation time for high molecular weight forms

• Targeted Antibody Approach:

  • Utilize epitope-specific antibodies targeting different regions of WDPCP

  • Compare detection patterns between antibodies recognizing:

    • N-terminal epitopes

    • C-terminal epitopes (such as the biotin-conjugated antibody targeting 621-737AA)

    • Internal domain-specific epitopes

  • Discrepancies in detection patterns can identify truncated or alternatively spliced variants

• Enzymatic Treatments:

  • Phosphatase Treatment: Incubate samples with lambda phosphatase to remove phosphate groups

    • Compare migration patterns before and after treatment

    • Merging of multiple bands into a single band indicates phosphorylation variants

  • Glycosidase Treatment: Apply PNGase F or Endo H to remove N-linked glycans

    • Shifts in molecular weight reveal glycosylated forms

  • Ubiquitin-Specific Proteases: Use to identify ubiquitinated WDPCP forms

• Mass Spectrometry Analysis:

  • Immunoprecipitate WDPCP using the biotin-conjugated antibody and streptavidin beads

  • Perform tryptic digestion of purified protein

  • Analyze peptide fragments by LC-MS/MS

  • Identify splice junctions and post-translational modifications

  • Quantify relative abundance of different forms

Data Analysis and Interpretation:

Experimental Design for Variant Analysis:

• Cell Type Comparison:

  • Process samples from different cell types known to express WDPCP

  • Run side-by-side on Western blots

  • Compare migration patterns to identify cell type-specific variants

  • Correlate with functional differences in MAPK signaling activation

• Stimulation Time Course:

  • Treat cells with stimuli known to activate WDPCP-related pathways

  • Collect samples at multiple time points (5, 15, 30, 60 minutes)

  • Analyze changes in the pattern of WDPCP forms

  • Correlate with downstream signaling events (MAPK activation)

• Subcellular Fractionation:

  • Separate cellular compartments (cytoplasm, membrane, nucleus)

  • Analyze distribution of WDPCP forms across fractions

  • Determine if specific variants localize to particular compartments

  • Correlate with functional roles in different cellular locations

• Functional Correlation:

  • Express specific WDPCP variants in WDPCP-knockout cells

  • Assess rescue of phenotypes (cell migration, EMT, MAPK activation)

  • Determine functional significance of each variant

By implementing these methodological approaches, researchers can effectively differentiate between WDPCP splice variants and post-translationally modified forms, providing deeper insights into the regulation and function of this important signaling protein.

What considerations are important when using WDPCP antibody, biotin conjugated in different experimental models?

Model-Specific Considerations for WDPCP Antibody Applications:

• Cell Culture Models:

  Human Cardiac Microvascular Endothelial Cells (HCMECs):

  • Optimal antibody dilution: 1:1000 for Western blot

  • Expected WDPCP expression: Moderate baseline levels

  • Key experimental conditions: Modulation by leucine levels demonstrated

  • Special considerations: Co-stain for EMT markers (E-cadherin, N-cadherin, vimentin) for comprehensive analysis

  Other Cell Lines:

  • Verify WDPCP expression levels before experiments (may vary widely)

  • Optimize antibody concentration for each cell type

  • Consider cell density effects on WDPCP expression

  • Account for serum factors that may influence WDPCP levels

• Animal Models:

  Mouse Models:

  • Species reactivity: The biotin-conjugated antibody is human-specific; verify cross-reactivity or use mouse-specific alternatives

  • Tissue preparation: Optimize fixation protocols for immunohistochemistry

  • Controls: Include WDPCP knockout tissues when available

  • Special considerations: Background signal in highly vascularized tissues may require additional blocking steps

  Disease Models (e.g., High-Leucine Diet Model):

  • Account for altered WDPCP expression in disease states

  • Include appropriate controls (untreated littermates)

  • Consider temporal dynamics of WDPCP expression during disease progression

  • Correlate with phenotypic measurements (e.g., epicardial adipose tissue volume)

• Human Clinical Samples:

  Tissue Sections:

  • Optimize antigen retrieval methods for formalin-fixed paraffin-embedded tissues

  • Account for autofluorescence in tissues like cardiac muscle

  • Consider batch effects between patient samples

  • Include normal adjacent tissue as internal control

  Plasma/Serum Samples:

  • Standardize collection and processing protocols

  • Account for diurnal variations in protein levels

  • Consider patient demographics and medication status

  • Use consistent protein quantification methods

Application-Specific Optimization Table:

Cross-System Validation Strategies:

• Correlation Across Models:

  • Compare WDPCP detection in cell lines vs. tissue samples

  • Validate findings from animal models in human specimens

  • Ensure consistency in molecular weight and detection patterns

  • Account for species-specific differences in WDPCP sequence and expression

• Methodological Triangulation:

  • Combine multiple detection methods (Western blot, immunofluorescence, ELISA)

  • Verify findings with orthogonal approaches (RT-PCR, RNA-seq)

  • Correlate protein detection with functional assays

  • Implement quantitative image analysis for tissue-based studies

• Scale Considerations:

  • Adjust protocols for sample size limitations (micro-methods for limited specimens)

  • Consider detection sensitivity requirements for different models

  • Adapt blocking and washing steps for tissue architecture

  • Optimize signal amplification for low-expression contexts

Special Adaptations for Challenging Models:

• 3D Organoid Cultures:

  • Increase antibody incubation time (24-48 hours)

  • Implement clearing techniques for better penetration

  • Use confocal microscopy for spatial resolution

  • Consider whole-mount immunostaining approaches

• Embryonic Tissues:

  • Account for developmental regulation of WDPCP expression

  • Adapt fixation to preserve delicate structures

  • Implement optical sectioning for 3D reconstruction

  • Consider co-staining with developmental markers

By carefully addressing these model-specific considerations, researchers can effectively apply WDPCP antibody, biotin conjugated across diverse experimental systems, ensuring reliable and comparable results that advance understanding of WDPCP biology and function.

What are the key takeaways for researchers planning to use WDPCP antibody, biotin conjugated in their studies?

Researchers planning to use WDPCP antibody, biotin conjugated should consider several key points to ensure successful experimental outcomes:

Experimental Design Guidelines:

• Antibody Characteristics Understanding:

  • Recognize that this antibody targets human WDPCP (UniProt: O95876), specifically amino acids 621-737

  • Remember it is a polyclonal antibody raised in rabbit, which offers broad epitope recognition but may have batch variation

  • Store appropriately at -20°C or -80°C and avoid repeated freeze-thaw cycles to maintain functionality

• Application-Specific Optimization:

  • Validated for ELISA applications according to manufacturer specifications

  • Western blot applications require optimization with 5μg protein loading and 1:1000 dilution as starting points

  • Immunofluorescence may require signal amplification strategies for optimal detection

  • Always include positive and negative controls specific to each application

• Biological Context Awareness:

  • WDPCP functions as an upstream regulator of MAPK/ERK signaling

  • Expression levels change in response to environmental factors (e.g., high leucine)

  • Role in cell migration and EMT processes makes it relevant for developmental and disease studies

  • Consider examining WDPCP in conjunction with its signaling partners and downstream effectors

Technical Implementation Checklist:

✓ Validate antibody specificity in your experimental system before conducting main experiments
✓ Optimize biotin-streptavidin detection system for your specific application
✓ Consider signal amplification methods for tissues or cells with low WDPCP expression
✓ Include appropriate controls (isotype, blocking peptide, WDPCP-depleted samples)
✓ Document lot number and prepare consistent protocols to ensure reproducibility
✓ Prepare for troubleshooting by understanding common challenges and solutions

Strategic Research Applications:

• WDPCP-MAPK Signaling Studies:

  • Use to investigate upstream regulation of MAPK pathway in developmental contexts

  • Combine with phospho-specific antibodies to correlate WDPCP levels with pathway activation

  • Consider kinase-catalyzed biotinylation approaches for comprehensive signaling analysis

• Cardiac Development Research:

  • Apply in studies of congenital heart defects and cardiac development

  • Explore WDPCP's role in epicardial EMT and cardiac cell migration

  • Investigate connections between WDPCP, cell polarity, and tissue architecture

• Cell Migration and EMT Investigations:

  • Implement in wound healing and transwell migration assays

  • Correlate WDPCP expression with EMT marker profiles

  • Explore interventions that modulate WDPCP levels and their functional outcomes

The biotin-conjugated format offers particular advantages for detection flexibility, signal amplification, and compatibility with various detection systems. By understanding the specific characteristics and applications of this tool, researchers can effectively incorporate it into their experimental workflows to advance understanding of WDPCP biology and its role in developmental and pathological processes.

What emerging research directions might benefit from WDPCP antibody, biotin conjugated applications?

Several promising research directions could significantly benefit from applications utilizing WDPCP antibody, biotin conjugated:

Developmental Biology and Congenital Disorders:

• Extended Bardet-Biedl Syndrome (BBS) Research:

  • WDPCP is also known as BBS15 protein, suggesting involvement in this ciliopathy

  • Investigation of WDPCP expression patterns during embryonic development

  • Correlation studies between WDPCP levels and BBS phenotypes

  • Exploration of potential therapeutic interventions targeting WDPCP pathways

• Planar Cell Polarity in Organogenesis:

  • Study of WDPCP's role in establishing tissue architecture beyond cardiac development

  • Investigation of epithelial organization in other organ systems

  • Correlation between WDPCP expression and ciliary function

  • Multi-organ developmental analyses in models with WDPCP manipulation

Cardiovascular Medicine:

• Personalized Medicine Approaches for CHDs:

  • Development of WDPCP expression profiling as a potential biomarker for CHD subtypes

  • Correlation between WDPCP variants and response to surgical interventions

  • Stratification of CHD patients based on WDPCP-related pathway activity

  • Design of targeted therapeutic approaches based on WDPCP status

• Cardiac Regeneration Studies:

  • Investigation of WDPCP in cardiac progenitor cell migration and differentiation

  • Exploration of WDPCP-mediated EMT in cardiac repair processes

  • Development of interventions to modulate WDPCP activity for enhancing regeneration

  • Correlation of WDPCP with epicardial activation after cardiac injury

Cell Biology Innovations:

• Mechanobiology Integration:

  • Studies connecting WDPCP function with mechanosensing in endothelial cells

  • Investigation of flow-responsive WDPCP regulation in vascular development

  • Correlation between matrix stiffness, WDPCP expression, and EMT processes

  • Development of biomaterial approaches incorporating WDPCP activity modulation

• Single-Cell Resolution Studies:

  • Application of biotin-conjugated antibody in mass cytometry (CyTOF) for high-dimensional analysis

  • Integration with single-cell transcriptomics for multi-omics approaches

  • Spatial transcriptomics correlation with WDPCP protein expression

  • Heterogeneity mapping of WDPCP activity in developing tissues

Methodological Advances:

• Proximity Labeling Applications:

  • Adaptation of the biotin-conjugated antibody for proximity labeling approaches

  • Identification of novel WDPCP interaction partners in different cellular contexts

  • Temporal mapping of WDPCP-associated complexes during differentiation

  • Spatial organization studies of WDPCP within cell polarity complexes

• Live Cell Imaging Strategies:

  • Development of cell-permeable detection systems for the biotin-conjugated antibody

  • Real-time visualization of WDPCP dynamics during cell migration

  • Correlation with cytoskeletal reorganization during polarization

  • Multiplex imaging with other cellular structures (Golgi, centrosome) relevant to polarity

Metabolism-Development Connections:

• Nutrient Sensing and Developmental Pathways:

  • Further exploration of leucine-WDPCP connections in developmental contexts

  • Investigation of other metabolic signals that may regulate WDPCP expression

  • Mechanistic studies linking maternal nutrition to offspring WDPCP levels

  • Development of dietary interventions for CHD prevention based on WDPCP pathway

• Adipose Tissue-Cardiac Interactions:

  • Detailed investigation of WDPCP's role in regulating epicardial adipose tissue

  • Exploration of adipocyte-cardiomyocyte signaling mediated by WDPCP

  • Correlation studies between WDPCP levels and cardiac adiposity in patient samples

  • Therapeutic targeting of WDPCP to modify epicardial adipose tissue in heart disease