WFIKKN1 Antibody

What is WFIKKN1 and why is it a significant research target?

WFIKKN1 (WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 1) is a multidomain secreted protein with significant research interest due to its role as a multivalent protease inhibitor. It contains a WAP domain, a follistatin domain, an immunoglobulin domain, two tandem Kunitz domains, and an NTR domain, suggesting it may control the action of multiple types of serine proteases and metalloproteinases . The protein is 548 amino acids in length with a molecular weight of approximately 58.8 kDa and belongs to the WFIKKN protein family . WFIKKN1 is particularly interesting to researchers studying muscle development, as data suggest that myostatin binding to its receptor (activin receptors type II) can be blocked by WFIKKN1 but not by the related protein WFIKKN2 .

What are the primary detection methods for WFIKKN1 in biological samples?

The primary methods for detecting WFIKKN1 in biological samples include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Sandwich ELISA techniques are widely used, with detection ranges typically between 31.2 pg/mL and 20 ng/mL depending on the kit . The technique employs antibodies specific for WFIKKN1 pre-coated onto a microplate, allowing quantitative measurement in serum, plasma, cell culture supernatants, and tissue homogenates .

• Western Blotting: Typically utilizing dilutions of 0.1-2 μg/mL of antibody, allowing for protein detection and semi-quantitative analysis .

• Immunohistochemistry: Using antibody concentrations around 25 μg/mL to visualize tissue distribution .

• Immunoprecipitation: For isolating WFIKKN1 protein from complex sample mixtures .

These methods can be optimized depending on the specific research question and sample type.

What tissue distribution pattern does WFIKKN1 exhibit?

WFIKKN1 shows a specific tissue distribution pattern. It is primarily expressed in:

• Pancreas

• Kidney

• Liver

• Placenta

• Lung

This tissue-specific expression suggests potentially specialized roles in these organs. The protein is known to be secreted and undergoes post-translational glycosylation, which may affect its functional properties in different tissue environments . Understanding this distribution pattern is crucial when designing experiments to study WFIKKN1 in specific physiological contexts.

How should WFIKKN1 ELISA protocols be optimized for maximum sensitivity?

To optimize WFIKKN1 ELISA protocols for maximum sensitivity:

• Sample Preparation: For serum and plasma samples, avoid repeated freeze-thaw cycles as they may degrade WFIKKN1. Process samples immediately after collection and store aliquots at -20°C or -80°C .

• Standard Curve Optimization: Create a reliable standard curve using purified WFIKKN1 protein. Most kits offer detection ranges between 0.312-20 ng/mL, with sensitivity as low as 0.078 ng/mL for human WFIKKN1 . For mouse WFIKKN1, detection ranges typically span 31.2-2000 pg/mL with a minimum detection limit of 31.2 pg/mL .

• Incubation Parameters: Follow the quantitative sandwich enzyme immunoassay technique where:

  • Initial binding occurs between sample and immobilized antibody

  • Biotin-conjugated detection antibody is added after washing

  • Streptavidin-HRP complex binds to the biotin-conjugated antibody

  • Substrate solution generates color proportional to WFIKKN1 concentration

• Antibody Selection: Choose antibodies with verified low cross-reactivity to related proteins to ensure specificity, particularly when studying both WFIKKN1 and WFIKKN2 in the same system .

• Validation Controls: Include positive controls (zebrafish, zebrafish head tissue, zebrafish body tissue for zebrafish studies; known positive human samples for human studies) to verify assay performance .

What are the critical considerations when selecting anti-WFIKKN1 antibodies for specific applications?

When selecting anti-WFIKKN1 antibodies for specific applications, researchers should consider:

• Target Species Compatibility: Ensure the antibody has been validated for your species of interest. Available options include antibodies reactive to human or zebrafish WFIKKN1 .

• Application-Specific Validation: Verify that the antibody has been tested and validated for your specific application:

  • For Western blotting: Antibodies with dilution ranges of 0.1-2 μg/mL

  • For immunohistochemistry: Antibodies validated at ~25 μg/mL

  • For immunoprecipitation: Antibodies validated at ~25 μg/mL

  • For ELISA: Antibodies optimized at 0.5-1 μg/mL

• Epitope Information: Consider the epitope region. For example, some antibodies target specific regions like AA 20-548 of human WFIKKN1 .

• Clonality Considerations:

  • Polyclonal antibodies may offer broader epitope recognition but potentially less specificity

  • Monoclonal antibodies provide consistent lots with high specificity to a single epitope

• Format Compatibility: Consider whether the antibody format (liquid, lyophilized) matches your storage capabilities and experimental workflow .

• Cross-Reactivity Profile: Review documentation on potential cross-reactivity with related proteins, especially WFIKKN2, which shares structural similarities with WFIKKN1 .

How does WFIKKN1 contribute to AHR signaling pathways and what methods best detect this interaction?

WFIKKN1 has been identified as a significant component in aryl hydrocarbon receptor (AHR) signaling pathways, particularly in zebrafish development. Research findings indicate:

• Co-expression Patterns: WFIKKN1 is highly co-expressed with CYP1A (a marker for AHR activation) in zebrafish exposed to polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) .

• Concentration-Dependent Expression: WFIKKN1 expression increases with TCDD concentration in a pattern parallel to CYP1A, though typically at lower expression levels, suggesting a dose-response relationship in AHR pathway activation .

• Temporal Expression Pattern: Similar to CYP1A, WFIKKN1 is not significantly induced at 24 hours post-fertilization (hpf) but shows significant induction by 48 hpf in zebrafish exposed to various chemicals .

For detecting this interaction, the following methods have proven effective:

• RNA Sequencing: To identify differential gene expression between wild-type and WFIKKN1 mutants after TCDD exposure .

• Mass Spectrometry-Based Proteomics: To identify differentially expressed proteins in response to WFIKKN1 mutation .

• CRISPR-Cas9 Mutation: Creating targeted mutations (e.g., 16-bp deletion in exon) to study functional changes in AHR signaling .

• qPCR Assays: To measure expression changes of WFIKKN1 alongside AHR pathway markers like CYP1A .

These experimental approaches have revealed that WFIKKN1 is involved in skeletal muscle development and plays a role in neurological pathways after TCDD exposure, making it a necessary functional member of the AHR signaling cascade.

What techniques can resolve contradictory WFIKKN1 expression data across different experimental systems?

When faced with contradictory WFIKKN1 expression data across different experimental systems, researchers can employ several strategies:

• Standardized Sample Processing:

  • Use consistent tissue/cell lysis buffers with appropriate protease inhibitors

  • Standardize protein quantification methods (BCA or Bradford assays)

  • Process all comparative samples simultaneously

• Multi-Method Validation:

  • Employ both protein-level (Western blot, ELISA) and mRNA-level (qPCR, RNA-seq) detection

  • Use ELISA kits with different epitope specificities to verify consistency

  • Confirm with immunohistochemistry for tissue distribution patterns

• Antibody Validation Controls:

  • Include WFIKKN1-knockout or knockdown samples as negative controls

  • Use recombinant WFIKKN1 protein as a positive control

  • Test multiple validated antibodies targeting different epitopes of WFIKKN1

• Species-Specific Considerations:

  • When comparing across species (e.g., human vs. zebrafish), use antibodies validated for each specific species

  • Consider potential evolutionary differences in WFIKKN1 structure and function

• Technical Approaches for Resolving Discrepancies:

  • Absolute quantification using mass spectrometry with isotope-labeled standards

  • Proximity ligation assays to verify protein-protein interactions in situ

  • Single-cell analysis techniques to address potential heterogeneity within samples

These approaches can help researchers determine whether contradictory data stem from biological variability or technical artifacts.

How can researchers distinguish between WFIKKN1 and WFIKKN2 in functional studies?

Distinguishing between WFIKKN1 and WFIKKN2 in functional studies requires careful experimental design:

• Selective Antibody Approaches:

  • Use antibodies that have been specifically validated for non-cross-reactivity between WFIKKN1 and WFIKKN2

  • Perform pre-absorption controls with recombinant proteins to verify specificity

  • Consider using epitope-specific antibodies targeting non-conserved regions

• Genetic Manipulation Strategies:

  • Employ CRISPR-Cas9 to create specific knockout models for each protein

  • Use siRNA or shRNA with validated specificity for selective knockdown

  • Employ rescue experiments with expression constructs containing mutations in key functional domains

• Functional Discrimination:

  • Exploit the differential binding of myostatin: research indicates that various forms of myostatin (promyostatin, myostatin pro-domain, and latent/processed myostatin) bind to WFIKKN1 but not WFIKKN2

  • Design binding assays that leverage this differential binding pattern

  • Use competitive binding experiments to distinguish between the two proteins

• Expression Pattern Analysis:

  • Analyze tissue-specific expression patterns, as WFIKKN1 and WFIKKN2 may have distinct tissue distributions

  • Perform dual immunostaining to identify areas of unique or overlapping expression

• Domain-Specific Functional Assays:

  • Design assays targeting the unique functional properties or binding partners of each protein

  • Measure protease inhibition specificity profiles, as WFIKKN1 and WFIKKN2 may inhibit different proteases with varying efficiencies

These approaches enable researchers to clearly distinguish the specific roles and functions of WFIKKN1 versus WFIKKN2 in their experimental systems.

What are the most common causes of false positives/negatives in WFIKKN1 detection assays?

Common causes of false results in WFIKKN1 detection assays include:

False Positives:

• Cross-reactivity: Antibodies may bind to structurally similar proteins, particularly WFIKKN2 or other proteins containing WAP, Kunitz, or immunoglobulin domains .

• Sample Contamination: Heterophilic antibodies in samples can bridge capture and detection antibodies in sandwich assays.

• Edge Effects: In ELISA plates, wells at the edges may yield higher readings due to uneven temperature distribution during incubation.

• Insufficient Blocking: Inadequate blocking can lead to non-specific binding of detection antibodies.

False Negatives:

• Protein Degradation: WFIKKN1 may degrade during improper sample handling or storage, as it is sensitive to repeated freeze-thaw cycles .

• Epitope Masking: Post-translational modifications like glycosylation may mask antibody binding sites .

• Matrix Effects: Components in biological samples may interfere with antibody binding.

• Detection Range Limitations: Sample concentrations below the minimum detection limit (e.g., 31.2 pg/mL for mouse WFIKKN1, 0.078 ng/mL for human WFIKKN1) .

Mitigation Strategies:

• Run appropriate positive and negative controls with each assay

• Perform spike-and-recovery experiments to assess matrix effects

• Use multiple antibodies targeting different epitopes to confirm results

• Validate findings using complementary methods (Western blot, mass spectrometry)

• Follow manufacturer's recommendations for sample handling and storage

How can researchers validate novel WFIKKN1 antibodies for research applications?

A comprehensive validation protocol for novel WFIKKN1 antibodies should include:

• Specificity Testing:

  • Western blot against recombinant WFIKKN1 protein to confirm expected molecular weight (58.8 kDa)

  • Testing against samples from WFIKKN1 knockout/knockdown models to confirm absence of signal

  • Cross-reactivity assessment with related proteins, particularly WFIKKN2

  • Peptide competition assays using the immunizing peptide

• Sensitivity Assessment:

  • Titration experiments to determine optimal antibody concentration for each application

  • Detection limit determination using serial dilutions of recombinant protein

  • Comparison with commercially validated antibodies

• Application-Specific Validation:

  • For Western blotting: Verify single band of appropriate molecular weight (58.8 kDa)

  • For IHC/ICC: Confirm expected tissue/cellular localization patterns

  • For IP: Demonstrate successful pull-down with mass spectrometry confirmation

  • For ELISA: Establish standard curve linearity and reproducibility

• Reproducibility Testing:

  • Inter-lab testing if possible

  • Lot-to-lot consistency assessment

  • Stability testing under various storage conditions

• Technical Documentation:

  • Detailed information on immunogen sequence and location within WFIKKN1

  • Host species, antibody isotype, and clonality

  • Optimal working dilutions for each validated application

  • Verified species reactivity

This validation framework ensures that novel antibodies are suitable for their intended research applications and will produce reliable, reproducible results.

What role does WFIKKN1 play in skeletal muscle development and how can it be investigated?

WFIKKN1 plays a significant role in skeletal muscle development, primarily through its interaction with myostatin signaling pathways. To investigate this role:

• Mechanistic Basis:

  • WFIKKN1 can block myostatin binding to its receptor (activin receptors type II), suggesting a regulatory role in muscle growth

  • Various forms of myostatin (promyostatin, myostatin pro-domain, and latent/processed myostatin) bind to WFIKKN1 but not WFIKKN2

  • Functional genomics studies in zebrafish have linked WFIKKN1 to muscle fiber development

• Investigative Approaches:

  • Genetic Manipulation: CRISPR-Cas9 knockout or knockdown models to assess muscle phenotypes in the absence of WFIKKN1

  • Protein Interaction Studies: Co-immunoprecipitation or proximity ligation assays to investigate WFIKKN1-myostatin interactions

  • Tissue-Specific Expression Analysis: Immunohistochemistry using validated antibodies (typically at 25 μg/mL) to examine WFIKKN1 distribution in developing muscle tissues

  • Functional Assays: Myoblast differentiation assays in the presence or absence of WFIKKN1 to assess effects on proliferation and differentiation

  • Proteomic Profiling: Mass spectrometry-based proteomics to identify differentially expressed proteins in WFIKKN1-deficient muscle tissue

• Experimental Models:

  • Zebrafish models have proven valuable for studying WFIKKN1 function in muscle development

  • Cell culture systems using C2C12 myoblasts or primary muscle cells can be employed for in vitro studies

  • Conditional knockout mouse models can provide insights into tissue-specific and developmental stage-specific roles

These approaches can help elucidate the precise molecular mechanisms by which WFIKKN1 influences muscle development and potentially identify therapeutic targets for muscle-related diseases.

How does WFIKKN1 interact with neurological pathways and what detection methods are most suitable for neural tissue?

WFIKKN1 has emerging roles in neurological pathways, particularly following environmental exposures such as TCDD. To study these interactions:

• Current Evidence:

  • Functional enrichment studies have demonstrated WFIKKN1 involvement in neurological pathways after TCDD exposure

  • WFIKKN1 mutant zebrafish exhibit significant behavior deficiencies at all life stages, suggesting neurological impacts

  • These findings indicate potential roles in neurodevelopment or neuroprotection

• Optimized Detection Methods for Neural Tissue:

  • Immunohistochemistry/Immunofluorescence: For localization in brain sections, using antibody concentrations of approximately 25 μg/mL

  • Western Blotting: Using 1-2 μg/mL antibody concentration for neural tissue lysates

  • In Situ Hybridization: To visualize mRNA expression patterns in specific neuronal populations

  • Single-Cell RNA Sequencing: To identify cell-type specific expression in heterogeneous neural tissues

  • Proximity Ligation Assays: To detect interactions between WFIKKN1 and neuronal proteins in situ

• Specialized Experimental Considerations:

  • Sample Preparation: Neural tissues require careful handling to preserve protein integrity; use of protease inhibitors and rapid processing is crucial

  • Background Reduction: Neural tissues often exhibit high background in immunostaining; extensive blocking and careful antibody titration are essential

  • Behavioral Assays: Correlate WFIKKN1 expression or mutation with specific behavioral phenotypes, as observed in zebrafish studies

  • Ex Vivo Systems: Organotypic brain slice cultures can be useful for studying WFIKKN1 function in intact neural circuits

These approaches enable the investigation of WFIKKN1's roles in normal neurodevelopment and its potential involvement in neurological disorders or responses to environmental toxicants.

What are the recommended controls for WFIKKN1 antibody validation in different experimental contexts?

Proper controls for WFIKKN1 antibody validation vary by experimental context:

For Western Blotting:

• Positive Control: Recombinant human WFIKKN1 protein or lysates from tissues known to express WFIKKN1 (pancreas, kidney, liver, placenta, lung)

• Negative Control: Lysates from WFIKKN1 knockout models or tissues known not to express WFIKKN1

• Antibody Controls: Primary antibody omission; isotype control antibody

• Loading Control: Probing for housekeeping proteins (β-actin, GAPDH)

• Molecular Weight Verification: Confirmation of the expected 58.8 kDa band

For Immunohistochemistry/Immunofluorescence:

• Positive Control Tissues: Pancreas, kidney, liver, placenta, lung sections

• Negative Control Tissues: Tissues from WFIKKN1 knockout models

• Antibody Controls: Primary antibody omission; isotype control antibody at matching concentration (25 μg/mL)

• Peptide Competition: Pre-incubation of antibody with immunizing peptide

• Counterstaining: Nuclear counterstain to assist in cellular localization interpretation

For ELISA:

• Standard Curve: Serial dilutions of recombinant WFIKKN1 protein

• Spike-and-Recovery: Known amounts of recombinant protein added to samples

• Dilutional Linearity: Serial dilutions of positive samples

• Blank Controls: Buffer-only wells to establish background

• Cross-Reactivity Controls: Testing related proteins, particularly WFIKKN2

For Zebrafish Studies:

• Positive Controls: Wild-type zebrafish head or body tissue

• Negative Controls: CRISPR-Cas9 WFIKKN1 mutant zebrafish (e.g., with 16-bp deletion in exon)

• Treatment Controls: Appropriate vehicle controls (e.g., dimethyl sulfoxide) for exposure experiments

These controls ensure the specificity, sensitivity, and reliability of WFIKKN1 detection across different experimental platforms.

What are the optimal storage conditions for maintaining WFIKKN1 antibody activity?

Optimal storage conditions for maintaining WFIKKN1 antibody activity vary by antibody format:

For Lyophilized Antibodies:

• Storage Temperature: Store at -20°C in a non-frost-free freezer

• Reconstitution: Use deionized water as specified in product documentation

• Post-Reconstitution: After reconstitution, store at 4°C for up to one month for active use

• Long-term Storage: For periods longer than one month, aliquot and store at -20°C to avoid repeated freeze-thaw cycles

• Avoid Contamination: Use sterile techniques during handling and aliquoting

For Liquid Antibodies:

• Short-term Storage (≤1 month): Store at 4°C

• Long-term Storage: Store at -20°C, with glycerol buffer to prevent freezing damage

• Buffer Conditions: Typically supplied in 50% glycerol, 0.9% NaCl, 0.2% Na₂HPO₄, 0.02% NaN₃

• Aliquoting: Divide into small working aliquots to minimize freeze-thaw cycles

• Temperature Transitions: Allow antibodies to reach room temperature before opening to prevent condensation

For ELISA Kits Containing WFIKKN1 Antibodies:

• Kit Storage: Store complete kits at 4°C until the expiration date, typically 6 months from production

• Component-Specific Storage: Some components may require -20°C storage; follow manufacturer's instructions

• Stability Considerations: Kit stability is typically determined by rate of activity loss, which should be less than 5% within the expiration date under appropriate storage conditions

• Working Solutions: Prepare fresh working solutions for each assay to maintain optimal activity

General Recommendations:

• Avoid Freeze-Thaw Cycles: Each cycle can decrease antibody activity by 5-20%

• Temperature Stability: Maintain consistent storage temperatures; avoid temperature fluctuations

• Light Protection: Store in amber tubes or wrapped in foil if the antibody is conjugated to light-sensitive fluorophores

• Documentation: Keep records of receipt date, lot number, and thawing/handling dates