WDPCP Antibody, HRP conjugated

What is WDPCP and why is it important in research?

WDPCP (WD repeat-containing and planar cell polarity effector protein fritz homolog) is a crucial regulator in developmental signaling pathways. It functions as an upstream modulator of MAPK/ERK signaling and plays essential roles in cardiac development. WDPCP influences cell migration and epithelial-mesenchymal transition (EMT) in human cardiac microvascular endothelial cells (HCMECs), making it a significant target in cardiac development research . The protein is also known by several aliases including hFRTZ, Bardet-Biedl syndrome 15 protein, and C2orf86 FRITZ . WDPCP's influence on cytoskeletal organization further contributes to its importance in developmental biology and cardiovascular research contexts .

What are the recommended storage conditions for WDPCP antibody, HRP conjugated?

For optimal stability and performance, WDPCP antibody with HRP conjugation should be stored at -20°C or -80°C upon receipt. It's crucial to avoid repeated freeze-thaw cycles as these can significantly degrade antibody performance . For HRP conjugates specifically, storing the antibody in undiluted form is strongly recommended whenever possible. While some researchers may prefer working concentrations for convenience, this approach can compromise long-term stability . The antibody is typically supplied in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during proper storage .

How should I verify the specificity of WDPCP antibody before experimental use?

To verify antibody specificity, perform western blot analysis using appropriate positive and negative controls. For WDPCP antibody, this would ideally include samples with known WDPCP expression levels as well as negative controls like knockout or knockdown samples . Evaluating band patterns for predicted size range (±20% of the expected molecular weight) provides initial validation. For enhanced validation, consider implementing approaches like:

• Genetic validation: siRNA knockdown to demonstrate decreased staining intensity

• Recombinant expression validation: overexpression systems showing increased signal

• Independent antibody validation: comparison with different antibodies targeting distinct epitopes

• Orthogonal validation: correlation with RNA expression data

• Protein array analysis: testing against panels containing 384 different antigens

What are the optimal conditions for using WDPCP antibody, HRP conjugated in western blot experiments?

For western blot applications using WDPCP antibody with HRP conjugation, follow these methodological guidelines based on established protocols:

• Protein extraction: Use RIPA buffer containing protease inhibitor cocktail

• Protein quantification: BCA assay for accurate loading (typically 5 μg protein per lane)

• Gel conditions: 12% polyacrylamide gel for optimal separation

• Transfer: To PVDF membrane using standard transfer protocols

• Blocking: 5% skimmed milk for 1 hour at room temperature

• Primary antibody dilution: Start with 1:1000 dilution in blocking buffer at 4°C for 24 hours

• Washing: PBS-T or TBS-T buffer, 3-5 washes of 5 minutes each

• Detection: Use western blot chemiluminescence kit

• Imaging: Capture using a GelDoc system or equivalent

• Analysis: Quantify band intensities using ImageJ, normalizing to β-actin as loading control

Optimization may be necessary for your specific experimental conditions, particularly regarding antibody dilution and incubation time.

How can I troubleshoot weak or absent signals when using WDPCP antibody, HRP conjugated?

When encountering weak or absent signals with WDPCP antibody, HRP conjugated, systematically evaluate these potential issues:

• Antibody concentration: Verify that the working dilution is appropriate; insufficient primary antibody can result in weak signals

• Antigen abundance: WDPCP may be expressed at low levels in your samples; consider loading more protein or using enrichment techniques

• Antigen accessibility: Ensure complete protein denaturation and sufficient membrane blocking

• Detection system sensitivity: Use high-sensitivity chemiluminescent substrates for low-abundance proteins

• HRP activity: Verify the activity of the HRP enzyme; improper storage or expired reagents can lead to reduced activity

• Transfer efficiency: Check protein transfer using Ponceau S or similar reversible stains

• Blocking conditions: Excessive blocking can mask epitopes; optimize blocking time and reagent

• Substrate incubation: Extend the substrate incubation time to allow for signal development

For particularly challenging samples, consider signal amplification systems designed for HRP-based detection methods.

How do I optimize WDPCP antibody, HRP conjugated for immunohistochemistry applications?

While the commercial WDPCP antibody with HRP conjugation may be primarily validated for ELISA, adapting it for immunohistochemistry requires careful optimization:

• Antigen retrieval: Test both heat-induced (citrate buffer, pH 6.0 or EDTA buffer, pH 9.0) and enzymatic methods to determine optimal epitope exposure

• Blocking endogenous peroxidase: Use 0.3-3% hydrogen peroxide for 10-15 minutes prior to antibody incubation

• Antibody titration: Perform dilution series (typically 1:100 to 1:1000) to identify optimal concentration

• Incubation conditions: Test both overnight at 4°C and 1-2 hours at room temperature

• Signal amplification: Consider tyramide signal amplification for low-abundance targets

• Counterstaining: Use appropriate nuclear counterstain (hematoxylin) that doesn't obscure specific signals

• Controls: Include positive and negative controls, including tissue known to express WDPCP and primary antibody omission controls

Document optimization steps thoroughly as tissue-specific conditions may vary considerably between experimental systems.

How can WDPCP antibody, HRP conjugated be used to investigate WDPCP/MAPK signaling in cardiac development?

WDPCP antibody with HRP conjugation provides a valuable tool for investigating the role of WDPCP in cardiac development through MAPK signaling pathways. Research has demonstrated that WDPCP acts as an upstream regulator of MAPK/ERK signaling, with WDPCP overexpression enhancing phospho-p38 and phospho-ERK levels . To investigate this pathway:

• Establish experimental models: Utilize cardiac microvascular endothelial cells, animal models with high-leucine diets, or genetic modification systems

• Manipulate WDPCP expression: Implement WDPCP overexpression or knockdown strategies

• Assess pathway components: Analyze both WDPCP and downstream MAPK components (phospho-p38, phospho-ERK) using western blotting with the HRP-conjugated antibody

• Functional assays: Measure cellular phenotypes including migration, invasion, and EMT markers (E-cadherin, N-cadherin, vimentin)

• Rescue experiments: Test whether MAPK activators like C16-PAF can rescue phenotypes in WDPCP-deficient systems

This approach allows for mechanistic dissection of how WDPCP regulates cardiac development through MAPK signaling cascades.

What controls should be included when using WDPCP antibody, HRP conjugated in western blot analysis of signaling pathways?

For rigorous analysis of WDPCP in signaling pathways using HRP-conjugated antibodies, include these essential controls:

• Positive control: Samples known to express WDPCP (specific cell lines or tissues)

• Negative control: Samples with WDPCP knockdown/knockout

• Loading control: β-actin or similar housekeeping protein to normalize expression levels

• Pathway modulation controls:

  • WDPCP overexpression samples

  • MAPK activator (e.g., C16-PAF) treated samples

  • Combined treatment samples

• Specificity control: Pre-absorption with immunizing peptide to confirm antibody specificity

• Technical controls: Secondary antibody-only control to assess non-specific binding

This comprehensive control strategy ensures reliable interpretation of results, particularly when investigating complex signaling relationships between WDPCP and MAPK pathway components.

How can I optimize multiplexed detection when using WDPCP antibody, HRP conjugated alongside other signaling proteins?

Multiplexed detection of WDPCP and related signaling proteins requires careful planning:

• Primary antibody selection: Choose antibodies raised in different host species to avoid cross-reactivity

• Sequential detection: For HRP-conjugated antibodies:

  • Complete first detection with WDPCP antibody, HRP conjugated

  • Document results thoroughly

  • Strip membrane using commercial stripping buffer (validate stripping efficiency)

  • Block again and reprobe with antibodies for other targets

• Fluorescent alternatives: Consider converting workflow to fluorescent secondaries for true multiplexing

• Sample preparation: Ensure extraction methods preserve all target proteins equally

• Data analysis: Use image analysis software that can accurately quantify multiple targets in relation to loading controls

For complex signaling pathway analysis, a combination of traditional western blotting with HRP-conjugated antibodies and phospho-specific flow cytometry may provide complementary insights.

What approaches can resolve contradictory results when using WDPCP antibody, HRP conjugated?

When faced with contradictory results using WDPCP antibody with HRP conjugation, implement this systematic troubleshooting approach:

• Antibody validation: Verify antibody specificity using enhanced validation protocols

  • siRNA knockdown

  • Recombinant expression

  • Independent antibodies targeting different epitopes

• Technical verification:

  • Test multiple lysate preparation methods

  • Vary blocking conditions

  • Adjust antibody concentrations

• Orthogonal approaches:

  • Complement protein detection with mRNA analysis

  • Utilize immunoprecipitation followed by mass spectrometry

• Biological considerations:

  • Assess cell type-specific expression patterns

  • Consider post-translational modifications affecting epitope recognition

  • Evaluate splice variants that might affect antibody binding

• Statistical analysis:

  • Increase biological replicates

  • Apply appropriate statistical tests for small sample sizes

This systematic approach helps identify whether contradictions stem from technical limitations or represent true biological complexity.

How should WDPCP antibody, HRP conjugated be used in studies of epithelial-mesenchymal transition?

For investigating WDPCP's role in epithelial-mesenchymal transition (EMT), design experiments that capture both molecular and functional aspects:

• Molecular markers: Use WDPCP antibody, HRP conjugated in western blot analysis alongside key EMT markers:

  • Epithelial markers: E-cadherin (typically 1:1500 dilution)

  • Mesenchymal markers: N-cadherin (1:1500) and vimentin (1:1000)

• Functional assays:

  • Migration assays: Transwell or wound healing

  • Invasion assays: Matrigel-coated chambers

• WDPCP manipulation strategies:

  • Overexpression with validated vectors

  • siRNA or shRNA knockdown

  • CRISPR/Cas9 gene editing

• Pathway analysis:

  • Monitor MAPK signaling components (phospho-p38, phospho-ERK)

  • Include pathway activators (C16-PAF) or inhibitors

• Quantification approach:

  • Normalize protein expression to loading controls

  • Establish clear criteria for morphological assessment

  • Use automated analysis software for migration/invasion quantification

This comprehensive approach provides mechanistic insights into how WDPCP regulates EMT processes.

What are the key considerations when selecting control tissues for WDPCP antibody validation?

Selecting appropriate control tissues for WDPCP antibody validation requires consideration of:

• Expression patterns: Choose tissues with known WDPCP expression levels

  • Positive controls: Heart tissue (particularly cardiac endothelial cells)

  • Negative/low expression controls: Tissues with minimal WDPCP expression

• Developmental stage: Consider embryonic versus adult tissues based on research context

• Species considerations:

  • Ensure species cross-reactivity if using non-human models

  • Verify epitope conservation across species

• Pathological samples:

  • Include relevant disease models (CHD models)

  • Match control tissues appropriately

• Technical controls:

  • Primary antibody omission

  • Isotype controls

  • Blocking peptide competition

Careful selection of control tissues enhances confidence in antibody specificity and experimental results.

How can WDPCP antibody, HRP conjugated be used to investigate protein-protein interactions in signaling complexes?

To investigate WDPCP interactions in signaling complexes:

• Co-immunoprecipitation (Co-IP):

  • Use non-HRP conjugated WDPCP antibody for immunoprecipitation

  • Validate pulled-down complexes using WDPCP antibody, HRP conjugated

  • Probe for interaction partners (MAPK pathway components)

• Proximity ligation assay (PLA):

  • Combine WDPCP antibody with antibodies against potential interaction partners

  • Visualize protein proximity in situ

• Bimolecular fluorescence complementation (BiFC):

  • Create fusion constructs of WDPCP and potential partners

  • Visualize interactions through reconstituted fluorescence

• FRET/FLIM analysis:

  • Use fluorescently labeled antibodies against WDPCP and interaction partners

  • Measure energy transfer as indication of proximity

• Validation approaches:

  • Confirm interactions under different cellular conditions

  • Test interactions with truncated protein domains

  • Verify with mass spectrometry approaches

These approaches provide complementary evidence for WDPCP's role in signaling complexes beyond traditional western blotting applications.

How can I optimize WDPCP antibody, HRP conjugated for detection in difficult tissue samples?

For challenging tissue samples:

• Fixation optimization:

  • Test multiple fixation protocols (PFA, methanol, acetone)

  • Adjust fixation duration to balance antigen preservation and accessibility

• Enhanced antigen retrieval:

  • Combine heat and enzymatic methods

  • Optimize pH conditions (acidic vs. basic buffers)

• Signal amplification systems:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems

• Background reduction:

  • Extended blocking (overnight at 4°C)

  • Inclusion of species-specific serum in blocking buffer

  • Pre-absorption of antibody with tissue powder

• Specialized extraction protocols:

  • Tissue-specific lysis buffers

  • Subcellular fractionation for enrichment

These approaches can significantly improve detection of WDPCP in samples that have proven challenging with standard protocols.

What are the best practices for quantitative analysis of WDPCP expression using HRP-conjugated antibodies?

For rigorous quantitative analysis:

• Standard curve generation:

  • Include recombinant WDPCP protein standards

  • Create multi-point calibration curves

• Linear detection range:

  • Determine the linear range of HRP detection for your system

  • Ensure samples fall within this range

• Normalization strategy:

  • Use validated housekeeping proteins (β-actin)

  • Consider multiple reference proteins for robust normalization

• Image acquisition:

  • Capture images below saturation threshold

  • Standardize exposure settings across experiments

• Analysis software:

  • Use dedicated quantification software (ImageJ, Image Lab)

  • Apply consistent analysis parameters

• Statistical considerations:

  • Perform technical and biological replicates

  • Apply appropriate statistical tests

  • Report variability measures

Adhering to these quantitative best practices enhances reproducibility and reliability of WDPCP expression data.

How do storage conditions affect the long-term performance of WDPCP antibody, HRP conjugated?

Storage conditions significantly impact antibody performance:

• Temperature effects:

  • Optimal storage at -20°C or -80°C for long-term stability

  • Avoid storage at 4°C for extended periods (>1 week)

• Freeze-thaw considerations:

  • Limit freeze-thaw cycles (<5 total)

  • Aliquot antibody upon first thaw

• Stabilizing additives:

  • Glycerol (50%) prevents freeze damage

  • Preservatives like Proclin 300 (0.03%) prevent microbial growth

• Dilution effects:

  • Store HRP conjugates undiluted when possible

  • Working dilutions have reduced stability

• Environmental factors:

  • Protect from light exposure

  • Minimize exposure to atmospheric oxygen

  • Avoid contamination with heavy metals that can affect HRP

• Monitoring stability:

  • Include reference samples in each experiment

  • Document signal intensity over time

Proper storage significantly extends the useful life of HRP-conjugated antibodies, maintaining sensitivity and specificity.