WFDC2 Recombinant Monoclonal Antibody

What is WFDC2 and what are its alternate nomenclatures in scientific literature?

WFDC2 is known by several names in scientific literature, including HE4 (Human Epididymis Protein 4), WAP5, EDDM4, dJ461P17.6, Epididymal secretory protein E4, Major epididymis-specific protein E4, and Putative protease inhibitor WAP5 . This protein functions as a broad-range protease inhibitor . Understanding these alternate names is crucial when conducting literature searches and comparing research findings across different publications, as some journals may preferentially use one nomenclature over others.

What are the molecular characteristics of WFDC2 protein?

The molecular weight of WFDC2 varies slightly depending on the detection method and protein form. In Western blotting applications, WFDC2 typically appears at approximately 13 kDa , though some sources report bands at around 20 kDa . For recombinant WFDC2 protein with a His tag, mass spectrometry analysis via ESI-TOF shows a predicted molecular weight of 11545.05 Da (±10 Da), with an observed weight of 11543.63 Da . The amino acid sequence of the mature protein typically spans from Thr28 to Phe124 , with the full functional sequence containing multiple cysteine residues that form the characteristic four-disulfide core domain.

What are the established research applications for WFDC2 antibodies?

WFDC2 antibodies are validated for multiple research applications, primarily:

• Enzyme-Linked Immunosorbent Assay (ELISA): Both as capture and detection antibodies in sandwich ELISA systems

• Western Blotting (WB): For protein detection in cell and tissue lysates

• Immunohistochemistry (IHC): For tissue section analysis

• Immunofluorescence (IF): For cellular localization studies

Each application requires specific antibody formats and optimization protocols to maximize sensitivity and specificity.

How should I select the appropriate WFDC2 antibody for my specific research application?

Selection of the appropriate WFDC2 antibody should be guided by your experimental requirements:

For ELISA development:

• Consider paired antibody systems where specific antibodies are designed to work together. For example, Mouse Anti-Human HE4/WFDC2 Monoclonal Antibody (such as Clone #676009) functions as an ELISA capture antibody when paired with Sheep Anti-Human HE4/WFDC2 Antigen Affinity-purified Polyclonal Antibody .

• For complex sample matrices, monoclonal antibodies typically offer higher specificity.

For Western Blotting:

• Rabbit polyclonal antibodies like the WFDC2 Antibody (600-401-FY9) have been validated for detecting the ~13 kDa band in human samples .

• Recombinant rabbit monoclonal antibodies like WFDC2 (E8S8Z) offer high specificity for detecting endogenous WFDC2 at approximately 20 kDa .

For immunostaining applications:

• Ensure the antibody has been specifically validated for IHC or IF as application performance can vary significantly .

What are the optimal conditions for using WFDC2 antibodies in Western blotting?

For optimal Western blotting with WFDC2 antibodies:

• Sample preparation:

  • Use appropriate lysis buffers that preserve protein structure while enabling efficient extraction

  • Include protease inhibitors to prevent degradation of WFDC2

• Electrophoresis conditions:

  • Use 12-15% SDS-PAGE gels to properly resolve the lower molecular weight WFDC2 protein (13-20 kDa)

  • Include positive controls such as recombinant WFDC2 protein

• Transfer and detection:

  • For WFDC2 (E8S8Z) Rabbit mAb, a recommended dilution of 1:1000 is appropriate

  • Expect bands at approximately 13 kDa or 20 kDa depending on the specific antibody and cell/tissue type

  • Secondary antibody selection should match the host species of primary antibody

• Validation strategies:

  • Include appropriate controls (positive, negative, loading controls)

  • Consider using different antibodies recognizing distinct epitopes to confirm specificity

How can I establish a reliable sandwich ELISA system for WFDC2 detection?

Developing a robust sandwich ELISA for WFDC2 requires careful consideration of antibody pairs and optimization:

• Antibody pair selection:

  • Use validated pairs like Mouse Anti-Human HE4/WFDC2 Monoclonal Antibody (MAB62741) as the capture antibody and Sheep Anti-Human HE4/WFDC2 Antigen Affinity-purified Polyclonal Antibody (AF6274) as the detection antibody

• Standard curve preparation:

  • Implement a 2-fold serial dilution of recombinant Human HE4/WFDC2 protein as demonstrated in the documented ELISA standard curves

  • Ensure standards span the expected concentration range in your samples

• Detection system optimization:

  • For biotinylated detection antibodies, use streptavidin-HRP conjugates with optimized incubation times

  • Appropriate substrate solutions (such as DY999) and stop solutions (such as DY994) should be used for consistent results

• Quantification:

  • Generate standard curves using appropriate curve-fitting models (typically 4PL or 5PL)

  • Ensure samples fall within the linear range of detection

How do different isoforms of WFDC2 affect antibody binding and experimental outcomes?

Multiple isoforms of WFDC2 are known to exist , which can impact antibody recognition and experimental results:

• Epitope considerations:

  • Antibodies targeting different regions may preferentially detect specific isoforms

  • The Rockland WFDC2 antibody (600-401-FY9) was produced using a 16 amino acid peptide near the N-terminus of human WFDC2 , while other antibodies may target different epitopes

• Experimental strategies:

  • Use antibodies raised against conserved regions to detect all isoforms

  • For isoform-specific detection, select antibodies targeting unique regions

  • Consider employing multiple antibodies recognizing different epitopes to comprehensively profile isoform expression

• Data interpretation:

  • Differences in band patterns or signal intensities between sample types may reflect differential isoform expression

  • Validate findings using complementary methods such as mass spectrometry or RT-PCR for isoform detection

What considerations are important when studying WFDC2 as a protease inhibitor?

When investigating the protease inhibitory function of WFDC2:

• Functional assays:

  • Design experiments to assess inhibition against a panel of proteases

  • Include positive controls (known inhibitors) and negative controls

  • Measure enzyme kinetics with varying concentrations of WFDC2

• Structural insights:

  • The WFDC domain contains eight cysteine residues forming four disulfide bonds

  • Mutations or modifications affecting these bonds may impact inhibitory activity

  • Consider how post-translational modifications may affect function

• Physiological relevance:

  • Correlate in vitro inhibition data with in vivo conditions

  • Consider the local concentration of WFDC2 in relevant tissues

  • Account for potential competitive or cooperative interactions with other inhibitors

What strategies can verify the specificity of WFDC2 antibodies in diverse experimental systems?

Comprehensive validation of WFDC2 antibody specificity should include:

• Multiple detection methods:

  • Cross-validate results using different techniques (WB, ELISA, IHC/IF)

  • Compare results from antibodies recognizing different epitopes

• Controls for specificity:

  • Recombinant WFDC2 protein as a positive control

  • Knockdown/knockout validation (siRNA, CRISPR)

  • Peptide competition assays using the immunizing peptide

  • Pre-absorption controls

• Cross-reactivity testing:

  • Test antibody performance across species if relevant

  • Evaluate potential cross-reactivity with related WAP domain-containing proteins

Why might I observe variable WFDC2 molecular weights across different detection methods?

Variations in observed molecular weight can occur due to several factors:

• Post-translational modifications:

  • Glycosylation can increase apparent molecular weight

  • Proteolytic processing can generate smaller fragments

  • Other modifications (phosphorylation, ubiquitination) may alter migration

• Technical factors:

  • Different gel systems (gradient vs. fixed percentage)

  • Running conditions (reducing vs. non-reducing)

  • Calibration of molecular weight markers

  • Recombinant vs. endogenous protein differences

• Recommendation table for troubleshooting molecular weight variations:

How can I address sensitivity limitations in WFDC2 detection protocols?

To enhance sensitivity in WFDC2 detection:

• For Western blotting:

  • Optimize antibody concentration (typically 1:1000 for WFDC2 antibodies)

  • Implement signal amplification methods (e.g., enhanced chemiluminescence)

  • Consider longer exposure times balanced against background

  • Use more sensitive detection systems (e.g., fluorescent secondary antibodies)

• For ELISA:

  • Optimize antibody pair selection using validated combinations

  • Increase sample incubation time at appropriate temperatures

  • Use signal amplification systems (e.g., poly-HRP conjugates)

  • Optimize blocking reagents to reduce background while preserving specific signal

• For immunostaining:

  • Implement antigen retrieval methods appropriate for WFDC2

  • Use tyramide signal amplification systems for low-abundance targets

  • Optimize primary antibody incubation (concentration, time, temperature)

  • Control for autofluorescence in IF applications

How should I interpret conflicting results between different WFDC2 antibody clones?

When facing discrepancies between different WFDC2 antibodies:

• Systematic comparison:

  • Document the exact experimental conditions used with each antibody

  • Compare epitope locations of different antibodies if known

  • Test antibodies side-by-side on identical samples

• Validation approaches:

  • Implement orthogonal detection methods (mRNA analysis, mass spectrometry)

  • Use genetic manipulation (overexpression, knockdown) to confirm specificity

  • Consider the possibility that different antibodies detect different isoforms or modified forms

• Application-specific considerations:

  • Some antibodies may perform better in certain applications

  • For example, the WFDC2 Rabbit Polyclonal Antibody (600-401-FY9) has been validated for ELISA, Western blotting, IHC, and IF , while others may have narrower application ranges