WFDC8 Antibody

What is WFDC8 and why is it significant in biological research?

WFDC8 belongs to the WAP-type four-disulfide core (WFDC) domain family. The WFDC domain contains eight cysteines that form four disulfide bonds at the protein's core, functioning primarily as a protease inhibitor. The WFDC8 protein contains a Kunitz-inhibitor domain and three WFDC domains, making it structurally unique within its family. Most WFDC genes are localized to chromosome 20q12-q13 in two clusters (centromeric and telomeric), with WFDC8 belonging to the telomeric cluster . Research interest in WFDC8 has increased due to its association with Kallmann Syndrome and its functional roles in protease inhibition, particularly through its serine-type endopeptidase inhibitor activity and peptidase inhibitor activity . SPINT1 is recognized as an important paralog of this gene, suggesting potential functional relationships worthy of investigation .

What types of WFDC8 antibodies are available for research applications?

Based on current research resources, multiple types of WFDC8 antibodies are available for different experimental applications. These primarily include polyclonal antibodies targeting the human WFDC8 protein. The Atlas Antibodies collection offers rabbit polyclonal anti-WFDC8 antibodies at 0.2 mg/ml concentration, designed specifically for human WFDC8 research . Abbexa also provides WFDC8 antibodies applicable for multiple applications and available in various quantities ranging from 20 μg to 1 mg . Most commercially available WFDC8 antibodies are non-conjugated, though this should be verified when selecting an antibody for specific applications . It's important to note that while there are numerous antibodies for other WFDC family members (particularly WFDC1 and WFDC2), specific WFDC8 antibodies are more limited but highly targeted for research applications.

What are the primary applications for WFDC8 antibodies in research?

WFDC8 antibodies support multiple research applications crucial for investigating this protein's expression, localization, and function. According to available sources, WFDC8 antibodies are validated for use in:

These applications enable researchers to investigate WFDC8 expression patterns across different tissues, subcellular localization, protein-protein interactions, and functional relationships with other proteins. Selecting the appropriate application depends on your specific research question and experimental design requirements.

How can I verify the specificity of a WFDC8 antibody before conducting experiments?

Antibody specificity verification is critical for generating reliable research data. For WFDC8 antibodies, consider implementing these validation approaches:

• Western blot analysis using positive and negative control samples (tissues/cells known to express or not express WFDC8)

• Immunoprecipitation followed by mass spectrometry confirmation

• RNA interference (siRNA or shRNA against WFDC8) to create knockdown controls

• Use of recombinant WFDC8 protein as a competitive inhibitor in your application

• Cross-validation with a second antibody targeting a different epitope of WFDC8

Many manufacturers provide validation data for their antibodies. For example, Atlas Antibodies states that their antibodies undergo rigorous validation in IHC, ICC-IF, and WB applications to ensure specificity and reproducibility . When selecting a WFDC8 antibody, examine the manufacturer's validation data and consider performing additional validation steps specific to your experimental system.

What are the optimal conditions for using WFDC8 antibodies in Western blotting protocols?

When using WFDC8 antibodies for Western blotting, consider these optimized methodological parameters:

Remember that these conditions should be optimized for your specific experimental setup and antibody source. Always perform preliminary experiments to determine the optimal conditions for your particular research context.

How can I troubleshoot weak or absent signal when using WFDC8 antibodies in immunohistochemistry?

When facing weak or absent signals in immunohistochemistry with WFDC8 antibodies, consider these methodological troubleshooting steps:

• Antigen retrieval optimization:

  • Try different antigen retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Extend retrieval time from standard 20 minutes to 30-40 minutes

  • Ensure complete cooling before proceeding to blocking step

• Antibody concentration adjustment:

  • Perform a titration experiment using different antibody dilutions (1:50, 1:100, 1:200, 1:500)

  • Extend primary antibody incubation time to overnight at 4°C instead of standard 1-2 hours

• Signal amplification strategies:

  • Implement tyramide signal amplification (TSA) for significantly enhanced sensitivity

  • Consider using polymer-based detection systems rather than standard ABC methods

• Tissue preparation considerations:

  • Ensure tissue fixation was appropriate (overfixation can mask epitopes)

  • Use freshly cut tissue sections (aged sections may have decreased antigenicity)

  • Validate the presence of the target using an alternative method (e.g., RT-PCR)

• Controls and validation:

  • Always include positive control tissue known to express WFDC8

  • Implement dual staining with a different WFDC8 antibody to confirm expression patterns

If signal remains problematic despite these optimizations, consider alternative detection methods such as RNA in situ hybridization to confirm expression patterns at the mRNA level before revisiting protein detection strategies.

What experimental considerations are important when investigating WFDC8 interactions with other proteins?

Investigating WFDC8 protein interactions requires careful experimental design. Consider these methodological approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use gentle lysis buffers (e.g., 0.5% NP-40) to preserve protein-protein interactions

  • Perform reciprocal Co-IPs (IP with anti-WFDC8 followed by Western blot for interaction partner, and vice versa)

  • Include appropriate negative controls (IgG control, lysates from cells with WFDC8 knockdown)

  • Consider crosslinking approaches for transient interactions

• Proximity ligation assays (PLA):

  • Effective for visualizing protein interactions in situ with single-molecule sensitivity

  • Requires two primary antibodies raised in different species (one for WFDC8, one for interaction partner)

  • Provides spatial information about where interactions occur within cells or tissues

• Bimolecular fluorescence complementation (BiFC):

  • Allows visualization of protein interactions in living cells

  • Requires genetic fusion of potential interaction partners with complementary fragments of a fluorescent protein

  • Particularly useful for dynamic studies of WFDC8 interactions

• Pull-down assays with recombinant proteins:

  • Use purified recombinant WFDC8 as bait protein

  • Can identify direct physical interactions without cellular context

  • Useful for confirming interactions identified through other methods

• Protease inhibition activity assays:

  • Since WFDC8 functions as a protease inhibitor, design functional assays measuring inhibition of candidate proteases

  • Use fluorogenic or chromogenic substrates to measure protease activity in presence/absence of WFDC8

  • Determine kinetic parameters (Ki values) to quantify interaction strength

When designing these experiments, consider that WFDC8 contains multiple domains, including three WFDC domains and a Kunitz-inhibitor domain , each potentially mediating different protein interactions. Domain-specific mutants can help identify which regions are responsible for specific interactions.

How should I design experiments to investigate WFDC8 expression patterns across different tissues?

Designing robust experiments to investigate WFDC8 expression patterns requires a multi-method approach:

• Tissue selection strategy:

  • Include tissues from multiple organ systems with emphasis on reproductive tissues, immune tissues, and epithelial surfaces

  • Consider developmental stages if investigating temporal expression patterns

  • Include tissues known to express other WFDC family members for comparative analysis

• Methodological approach combination:

  • Transcriptional analysis: qRT-PCR with validated WFDC8-specific primers and appropriate reference genes

  • Protein localization: Immunohistochemistry using validated anti-WFDC8 antibodies

  • Protein quantification: Western blot and/or ELISA for tissue lysates

• Validation through orthogonal methods:

  • RNA-seq for comprehensive transcriptional profiling

  • In situ hybridization to confirm cellular localization of mRNA

  • Mass spectrometry for unbiased protein detection and quantification

• Controls and standards implementation:

  • Include tissues from WFDC8 knockout models (if available) as negative controls

  • Use recombinant WFDC8 protein as positive control for antibody-based methods

  • Apply consistent quantification standards across different tissue types

• Data normalization approach:

  • Normalize protein expression to total protein content rather than single housekeeping proteins

  • Use multiple reference genes for qRT-PCR normalization

  • Apply appropriate statistical analysis for comparing expression across tissues

By implementing this comprehensive approach, you can generate reliable data on WFDC8 tissue distribution patterns that accounts for potential methodological limitations of any single technique.

What controls are essential when validating WFDC8 knockdown or overexpression in functional studies?

Rigorous controls are essential for validating WFDC8 manipulation in functional studies:

Additionally, implement these validation strategies:

• Expression level verification:

  • Confirm knockdown/overexpression at both mRNA level (qRT-PCR) and protein level (Western blot using validated antibodies )

  • Quantify and report percentage of knockdown/fold overexpression

• Functional validation:

  • Assess known WFDC8 functions (protease inhibition) using biochemical assays

  • Compare results with established phenotypes of other WFDC family members

• Specificity confirmation:

  • Assess expression of closely related family members (WFDC1, WFDC2) to rule out compensatory mechanisms

  • Evaluate expression of SPINT1 (important paralog ) after WFDC8 manipulation

• Cellular localization monitoring:

  • Use immunofluorescence with validated antibodies to confirm correct localization of overexpressed protein

  • Include tagged versions (GFP/FLAG) to monitor expression while verifying tag doesn't disrupt function

These controls ensure that observed phenotypes are specifically attributable to WFDC8 modulation rather than experimental artifacts or off-target effects.

How can I differentiate between functional effects of WFDC8 and other WFDC family members in my experimental system?

Differentiating functional effects of WFDC8 from other WFDC family members requires careful experimental design:

• Domain-specific functional analysis:

  • Unlike some WFDC family members, WFDC8 contains both WFDC domains (three) and a Kunitz-inhibitor domain

  • Create domain deletion mutants to identify contributions of specific domains

  • Compare inhibitory profiles against different proteases to identify WFDC8-specific targets

• Selective targeting strategies:

  • Design highly specific siRNAs/shRNAs targeting unique regions of WFDC8 mRNA

  • Utilize CRISPR/Cas9 genome editing for complete WFDC8 knockout

  • Implement rescue experiments with WFDC8 constructs resistant to siRNA but maintaining function

• Comparative expression analysis:

  • Perform parallel expression analysis of multiple WFDC family members

  • Create expression correlation matrices across different experimental conditions

  • Use hierarchical clustering to identify conditions where WFDC8 behaves distinctly from other family members

• Protease inhibition profile characterization:

  • Generate a comprehensive inhibition profile for WFDC8 against a panel of proteases

  • Compare with published profiles of other WFDC proteins

  • Identify proteases uniquely or preferentially inhibited by WFDC8

• Protein interaction network mapping:

  • Perform immunoprecipitation followed by mass spectrometry (IP-MS) to identify WFDC8-specific interaction partners

  • Compare with interaction networks of other WFDC family members

  • Focus functional studies on pathways uniquely regulated by WFDC8

By implementing these approaches, you can establish functional specificities of WFDC8 distinct from other family members, leading to more precise understanding of its biological roles.

What approaches can determine if post-translational modifications affect WFDC8 antibody recognition and protein function?

Post-translational modifications (PTMs) can significantly impact both antibody recognition and biological function of WFDC8. Consider these methodological approaches:

• PTM identification strategies:

  • Mass spectrometry analysis of immunoprecipitated endogenous WFDC8

  • Phospho-specific antibody screening if phosphorylation is suspected

  • Glycosylation analysis using glycosidase treatments followed by mobility shift assessment

  • Ubiquitination studies using ubiquitin-specific antibodies or tandem ubiquitin binding entities (TUBEs)

• Epitope mapping protocols:

  • Generate a panel of WFDC8 fragments to determine antibody binding regions

  • Create site-directed mutants of predicted PTM sites and assess antibody binding

  • Perform peptide competition assays with modified and unmodified peptides

• Functional impact assessment:

  • Compare activity of native versus deglycosylated/dephosphorylated WFDC8

  • Generate non-modifiable mutants (e.g., S/T→A for phosphorylation, N→Q for N-glycosylation)

  • Analyze subcellular localization of PTM-deficient mutants versus wild-type protein

• Antibody selection strategy for PTM research:

  • Choose antibodies raised against recombinant proteins (likely lacking PTMs) versus those against synthetic peptides

  • Validate antibody recognition using in vitro modified recombinant proteins

  • Consider using multiple antibodies targeting different epitopes when studying heavily modified proteins

• Temporal regulation investigation:

  • Assess PTM patterns under different cellular conditions (stress, differentiation, etc.)

  • Correlate changes in PTM status with functional activities

  • Identify enzymes responsible for adding/removing specific modifications

These approaches will help determine whether PTMs are critical for WFDC8 function and whether they influence experimental outcomes due to differential antibody recognition.

How can I design experiments to investigate potential roles of WFDC8 in inflammatory processes or immune regulation?

Investigating WFDC8's role in inflammatory and immune processes requires multifaceted experimental design:

• Expression regulation in inflammatory contexts:

  • Assess WFDC8 expression in immune cells (macrophages, neutrophils, epithelial cells) after stimulation with:

    • Pathogen-associated molecular patterns (PAMPs): LPS, poly(I:C), flagellin

    • Pro-inflammatory cytokines: TNF-α, IL-1β, IL-6

    • Anti-inflammatory cytokines: IL-10, TGF-β

  • Perform time-course and dose-response experiments

  • Use Western blot with validated antibodies and qRT-PCR for quantification

• Functional assessment in inflammatory models:

  • In vitro models:

    • WFDC8 overexpression/knockdown in macrophages followed by cytokine profiling

    • Neutrophil extracellular trap (NET) formation assays with recombinant WFDC8

    • Bacterial killing assays in presence/absence of WFDC8

  • Ex vivo models:

    • Precision-cut tissue slices treated with recombinant WFDC8

    • Primary cell cultures from WFDC8 knockout models (if available)

  • In vivo models:

    • Assess local and systemic inflammation in WFDC8-deficient animal models

    • LPS challenge studies with WFDC8 administration

• Mechanistic investigations:

  • Identify specific proteases inhibited by WFDC8 in inflammatory contexts

  • Investigate potential non-protease inhibitory functions (similar to other WFDC family members)

  • Assess impact of WFDC8 on inflammatory signaling pathways (NF-κB, MAPK, etc.)

• Translational relevance assessment:

  • Analyze WFDC8 levels in patient samples from inflammatory conditions

  • Correlate WFDC8 expression with disease severity or outcomes

  • Compare with known inflammatory biomarkers

This comprehensive approach will help elucidate whether WFDC8 has significant immunomodulatory functions similar to other WFDC family members while establishing its unique contributions to immune regulation.

What methodological approaches can evaluate the potential role of WFDC8 in cancer progression or suppression?

Investigating WFDC8's role in cancer requires systematic experimental approaches:

• Expression profile characterization:

  • Analyze WFDC8 expression across cancer types using:

    • Immunohistochemistry with validated antibodies on tissue microarrays

    • Western blot analysis of tumor vs. normal tissue lysates

    • Mining public databases (TCGA, GTEx, CCLE) for mRNA expression patterns

  • Correlate expression with clinicopathological parameters and patient outcomes

• Functional assessment in cancer models:

  • In vitro functional assays after WFDC8 modulation:

    • Proliferation assays (MTT, BrdU incorporation)

    • Migration and invasion assays (Transwell, wound healing)

    • Colony formation and soft agar growth

    • Apoptosis assessment (Annexin V, TUNEL)

  • In vivo tumor models:

    • Xenograft studies with WFDC8-overexpressing or WFDC8-knockdown cancer cells

    • Patient-derived xenografts treated with recombinant WFDC8

    • Metastasis models to assess impact on cancer dissemination

• Mechanistic investigations:

  • Protease regulation:

    • Identify cancer-relevant proteases inhibited by WFDC8

    • Focus on proteases involved in matrix remodeling and metastasis

  • Signaling pathway analysis:

    • Assess impact on growth factor signaling (EGFR, FGFR, etc.)

    • Investigate effects on PI3K/AKT, MAPK, and WNT pathways

    • Study potential interaction with tumor suppressor pathways

  • Tumor microenvironment effects:

    • Evaluate impact on tumor-associated macrophage function

    • Assess effects on angiogenesis and lymphangiogenesis

    • Analyze influence on immune cell infiltration and function

• Comparative analysis with other WFDC family members:

  • Compare WFDC8 cancer-related functions with WFDC1 (ps20, a known tumor suppressor)

  • Contrast with WFDC2 (HE4, a known oncogene and ovarian cancer biomarker)

  • Develop integrated models of WFDC family function in cancer contexts

This multifaceted approach will establish whether WFDC8 functions primarily as a tumor suppressor (like WFDC1) or promotes cancer progression (like WFDC2), informing its potential as a therapeutic target or biomarker.

How can I address cross-reactivity issues when using WFDC8 antibodies in tissues expressing multiple WFDC family members?

Addressing cross-reactivity concerns with WFDC8 antibodies requires systematic validation and experimental design:

• Comprehensive specificity testing:

  • Test antibodies against recombinant proteins of all WFDC family members

  • Perform Western blots using lysates from cells overexpressing individual WFDC proteins

  • Conduct peptide blocking experiments with specific peptides from WFDC8 and closely related family members

  • Implement knockout/knockdown controls for WFDC8 and related proteins

• Epitope analysis and selection:

  • Select antibodies targeting unique regions of WFDC8 with minimal sequence homology to other WFDC proteins

  • Analyze epitope sequences using multiple sequence alignment tools

  • Consider custom antibody development against highly specific WFDC8 regions if commercial options show cross-reactivity

• Application-specific validation protocols:

  • For immunohistochemistry: Perform parallel staining with multiple antibodies targeting different WFDC8 epitopes

  • For Western blotting: Use precise molecular weight differentiation and include positive controls

  • For immunoprecipitation: Validate pulled-down proteins by mass spectrometry

• Combined detection approaches:

  • Implement dual-labeling approaches (e.g., IF for protein combined with FISH for mRNA)

  • Correlate antibody staining patterns with known mRNA expression data from RNA-seq or qRT-PCR

  • Use proximity ligation assay (PLA) with two different WFDC8 antibodies for highly specific detection

• Data interpretation guidelines:

  • Establish clear criteria for positive WFDC8 staining based on validated controls

  • Document potential cross-reactivity in your experimental reports

  • Consider multiple methods to confirm key findings

By implementing these rigorous approaches, you can minimize cross-reactivity concerns and generate reliable data on WFDC8 expression and function in complex biological systems.

What are the optimal storage and handling conditions to maintain WFDC8 antibody performance over time?

Proper storage and handling of WFDC8 antibodies is critical for maintaining their performance and extending their useful lifespan:

• Storage temperature recommendations:

  • Store antibody aliquots at -20°C for long-term storage

  • Keep working aliquots at 4°C for up to 2 weeks

  • Avoid repeated freeze-thaw cycles (create single-use aliquots)

  • Some antibody formats may have specific manufacturer recommendations that supersede these general guidelines

• Aliquoting protocol:

  • Create small, single-experiment aliquots upon receiving the antibody

  • Use sterile conditions during aliquoting to prevent contamination

  • Include carrier protein (0.1% BSA) if diluting before aliquoting

  • Label tubes with antibody details, concentration, and date

• Buffer considerations:

  • Maintain recommended buffer conditions (typically PBS with preservative)

  • Consider adding sodium azide (0.02%) to prevent microbial growth

  • Some applications may require azide-free antibody preparations

• Handling precautions:

  • Minimize exposure to light for fluorophore-conjugated antibodies

  • Avoid vortexing antibodies; mix by gentle inversion or pipetting

  • Use clean, nuclease-free tubes for storage

  • Handle at appropriate temperatures (cold room or on ice)

• Performance monitoring strategy:

  • Include positive controls in each experiment to monitor antibody performance

  • Document lot numbers and maintain records of performance

  • Consider implementing a validation protocol for new lots

  • Track signal intensity over time to detect potential degradation

• Shipping and temporary storage considerations:

  • Use ice packs for short-term transport

  • For longer shipping, use dry ice

  • Upon receipt, immediately transfer to appropriate long-term storage

Following these guidelines will help ensure consistent performance of WFDC8 antibodies across experiments and extend their functional lifespan, improving experimental reproducibility and reducing costs associated with antibody replacement.

How should I approach contradictory results between different detection methods when studying WFDC8 expression?

When facing contradictory results between different methods of WFDC8 detection, implement this systematic troubleshooting approach:

• Method-specific limitations assessment:

  • Antibody-based methods: Evaluate specificity, sensitivity, and potential cross-reactivity issues

  • mRNA detection: Consider post-transcriptional regulation differences between mRNA and protein levels

  • Mass spectrometry: Assess sample preparation compatibility with WFDC8 detection

  • Document technical parameters that might affect detection (fixation methods, extraction protocols)

• Sample-specific considerations:

  • Evaluate tissue/cell heterogeneity that might explain discrepancies

  • Consider subcellular localization differences that might affect detection by different methods

  • Assess potential post-translational modifications affecting antibody recognition

  • Evaluate potential context-dependent expression differences in your experimental system

• Validation through orthogonal approaches:

  • Implement at least three independent detection methods:

    • Protein level: Western blot, immunohistochemistry, ELISA

    • mRNA level: qRT-PCR, RNA-seq, in situ hybridization

    • Functional approaches: Activity assays specific to WFDC8 function

  • Use genetic manipulation (overexpression/knockdown) to create defined controls

• Data integration and reconciliation strategy:

  • Create a comprehensive table documenting all results and methodological details

  • Identify patterns in discrepancies (e.g., consistent issues with particular methods)

  • Weight evidence based on methodological strengths and limitations

  • Consider biological explanations for apparent contradictions:

    • Alternative splicing affecting epitope availability

    • Tissue-specific post-translational modifications

    • Context-dependent protein stability or turnover

• External validation resources:

  • Compare your findings with published literature and public databases

  • Consult with experts in WFDC protein biology or specific methodologies

  • Consider collaborative verification of key findings using complementary expertise

Through this systematic approach, you can resolve contradictions between methods and develop a more accurate understanding of WFDC8 expression in your experimental system. In publications, transparently report discrepancies and provide your interpretation of these differences rather than selectively reporting concordant results.