wfikkn Antibody

What are WFIKKN proteins and why are they significant in research?

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins with complex structure and diverse biological functions. They derive their name from their domain composition: WAP domain, Follistatin domain, Immunoglobulin domain, two Kunitz-type protease inhibitor domains, and an NTR domain. These proteins have gained significant research interest due to their role in regulating various growth factors, particularly those in the TGFβ family. Their most notable function is serving as antagonists of growth and differentiation factors GDF8 (myostatin) and GDF11, while also binding (but not inhibiting) TGFβ1, BMP2, and BMP4 with relatively high affinity. This dual functionality makes them important regulators of skeletal muscle development and potential therapeutic targets .

What are the structural differences between WFIKKN1 and WFIKKN2?

Both WFIKKN1 and WFIKKN2 share the same domain architecture (WAP, follistatin, immunoglobulin, Kunitz, and netrin domains), but they differ in their primary sequence and tissue distribution. WFIKKN2 (also known as GASP-1) consists of 576 amino acids (Leu35-His576 in the mature protein) and is 90% identical to mouse GASP-1. Human WFIKKN1 and WFIKKN2 share approximately 55% sequence identity, suggesting divergent evolutionary roles. The antibodies targeting these proteins typically recognize specific epitopes within their structures - for example, the WFIKKN1 antibody ABIN204219 targets amino acids 20-548 of the human protein .

How do WFIKKN proteins interact with TGF-β family members?

WFIKKN proteins exhibit differential binding and inhibitory activities toward TGF-β family members. Surface plasmon resonance (SPR) measurements have demonstrated that both WFIKKN1 and WFIKKN2:

• Bind to GDF8 and GDF11 with high affinity and inhibit their biological activity in the nanomolar range

• Bind to TGFβ1, BMP2, and BMP4 with relatively high affinity (Kd ~10^-6 M) but do not inhibit their signaling activity even at micromolar concentrations

This selective inhibition pattern indicates that WFIKKN proteins function as true antagonists for GDF8 and GDF11, but act as growth factor binding proteins for TGFβ1, BMP2, and BMP4, potentially helping to establish growth factor gradients in the extracellular space .

What are the optimal protocols for using WFIKKN antibodies in Western blot applications?

When using WFIKKN antibodies for Western blot applications, researchers should follow these methodological guidelines:

• Sample preparation: Tissue homogenates or cell lysates should be prepared with protease inhibitors to prevent degradation of WFIKKN proteins

• Protein separation: Use 7-10% SDS-PAGE gels due to the large size of WFIKKN proteins (~65-70 kDa)

• Transfer conditions: Wet transfer at 30V overnight at 4°C is recommended for complete transfer of large proteins

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation: Optimal dilutions should be determined empirically for each application, but typical starting dilutions are 1:1000 for monoclonal antibodies like ABIN204219

• Detection: Standard HRP-conjugated secondary antibodies followed by ECL detection

For GASP-1/WFIKKN2 antibodies like AF2070, validation studies have confirmed successful application in Western blot analysis of mouse tissue homogenates, as referenced in published research on GASP-1 overexpressing mice .

What are the proper storage and handling conditions for WFIKKN antibodies?

To maintain optimal activity of WFIKKN antibodies, follow these storage and handling recommendations:

It is crucial to avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thawing. Use a manual defrost freezer rather than auto-defrost to prevent temperature fluctuations. For antibodies like WFIKKN2/GASP-1 (AF2070), reconstitution should be performed according to manufacturer instructions, typically with sterile PBS or similar buffer .

How can researchers validate the specificity of WFIKKN antibodies?

Validation of WFIKKN antibody specificity is critical for experimental reliability. Recommended validation approaches include:

• Positive controls: Use tissues known to express WFIKKN proteins (e.g., brain, skeletal muscle, and kidney for WFIKKN1; ovary, testis, and brain for WFIKKN2/GASP-1)

• Negative controls: Use tissues with minimal expression (or knockout samples where available)

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

• Recombinant protein controls: Test antibody against purified recombinant WFIKKN proteins

• Multiple antibody validation: Use different antibodies targeting different epitopes to confirm results

• Expression correlation: Correlate protein detection with mRNA expression data

These approaches ensure that signals detected in experimental applications genuinely represent the WFIKKN proteins of interest and not non-specific binding .

How can SPR measurements be optimized to study WFIKKN interactions with TGF-β family members?

Surface plasmon resonance (SPR) is a valuable technique for studying WFIKKN protein interactions with TGF-β family members. To optimize SPR experiments:

• Protein immobilization: Immobilize purified WFIKKN proteins on CM5 sensor chips using amine coupling chemistry at pH 4.5

• Analyte preparation: Prepare growth factors (GDF8, GDF11, TGFβ1, BMP2, BMP4) in running buffer with 0.005% surfactant

• Concentration series: Use a wide concentration range (1 nM to 1 μM) to accurately determine binding kinetics

• Flow rate: Maintain constant flow (30 μL/min) to minimize mass transport effects

• Regeneration: Use 10 mM glycine-HCl (pH 1.5) for surface regeneration between analyte injections

• Data analysis: Apply appropriate binding models (1:1 Langmuir binding, heterogeneous ligand) to calculate association and dissociation rate constants

This approach has successfully demonstrated the differential binding affinities of WFIKKN proteins to various TGF-β family members, revealing Kd values in the nanomolar range for GDF8/GDF11 and micromolar range for TGFβ1, BMP2, and BMP4 .

What experimental approaches can distinguish between the antagonistic and binding functions of WFIKKN proteins?

To differentiate between the antagonistic and binding functions of WFIKKN proteins, researchers should employ a combination of binding assays and functional reporter systems:

• Binding assays (SPR, co-immunoprecipitation) to establish physical interaction

• Reporter assays using cells transfected with:

  • SMAD-responsive luciferase reporters for BMP/GDF signaling

  • pGL3-(CAGA)12 for TGF-β signaling

The experimental design should include:

• Concentration-dependent response curves for each growth factor

• Pre-incubation of growth factors with varying concentrations of WFIKKN proteins

• Appropriate positive controls (known inhibitors) and negative controls

Analysis of dose-response curves can reveal whether WFIKKN proteins cause:

• Complete inhibition (rightward shift of the entire curve) - antagonistic function

• No inhibition despite binding (curve unchanged) - binding function without antagonism

This combined approach has demonstrated that WFIKKN proteins inhibit GDF8 and GDF11 in the nanomolar range while failing to inhibit TGFβ1, BMP2, and BMP4 even at micromolar concentrations despite physical binding .

What role do WFIKKN proteins play in muscle development and homeostasis?

WFIKKN proteins, particularly WFIKKN2/GASP-1, play crucial roles in muscle development and homeostasis through their interaction with myostatin (GDF8):

• Myostatin regulation: WFIKKN2/GASP-1 binds and inhibits myostatin, a negative regulator of skeletal muscle mass

• Dual binding capability: GASP-1 can bind both mature myostatin and the myostatin propeptide independently

• Muscle hypertrophy: Overexpression of GASP-1 in mice leads primarily to a hypermuscular phenotype

• Metabolic effects: GASP-1 overexpression also affects adiposity and glucose homeostasis in adult mice

Research approaches to study these effects include:

• Transgenic mouse models with tissue-specific WFIKKN2/GASP-1 overexpression

• Knockout models to assess loss-of-function phenotypes

• Muscle cell culture (C2C12 myoblasts) to study effects on differentiation

• Glycosylation studies to understand post-translational regulation

These methodologies have revealed that WFIKKN proteins are integral to maintaining proper muscle growth and metabolism, with potential therapeutic implications for muscle wasting disorders .

What are common issues when using WFIKKN antibodies in immunological techniques?

Researchers may encounter several challenges when working with WFIKKN antibodies:

• Cross-reactivity: Due to the 55% sequence identity between WFIKKN1 and WFIKKN2, antibodies may cross-react. Solution: Use epitope-mapped antibodies specifically validated for selectivity.

• Low signal intensity: WFIKKN proteins may be expressed at low levels in some tissues. Solution: Employ signal amplification methods (TSA, polymer detection systems) or increase protein loading.

• Background signal: Multiple domains in WFIKKN proteins can contribute to non-specific binding. Solution: Optimize blocking conditions (5% BSA may be more effective than milk for some applications) and include additional washing steps.

• Post-translational modifications: N-glycosylation of WFIKKN proteins may affect antibody recognition. Solution: Consider deglycosylation treatments if detecting native protein is problematic.

• Epitope masking: Protein-protein interactions may hide antibody epitopes. Solution: Test different extraction/lysis buffers to disrupt protein complexes.

Published research has shown that N-glycosylation of murine GASP-1 affects its secretion but not its activity on C2C12 myogenic cells, highlighting the importance of considering these modifications in experimental design .

How can researchers reconcile contradictory findings related to WFIKKN antibody applications?

When facing contradictory results with WFIKKN antibodies across different studies, consider the following methodological approaches:

• Antibody characterization: Different antibodies may target different epitopes, affecting detection of protein variants or modified forms

• Expression context: WFIKKN expression varies significantly by tissue type and developmental stage:

  • WFIKKN1: Highest in brain, skeletal muscle, thymus, kidney (fetal); ovary, testis, brain (adult)

  • WFIKKN2: Abundant in lung, skeletal muscle, liver (fetal); pancreas, liver, thymus (adult)

• Experimental models: Different cell lines or animal models may show distinct WFIKKN regulation patterns

• Technical variables: Standardize protocols for:

  • Antibody concentration and incubation time

  • Sample preparation methods

  • Detection systems

  • Quantification approaches

• Multi-method verification: Combine antibody-based techniques with:

  • RNA analysis (qPCR, RNA-seq)

  • Mass spectrometry

  • CRISPR-based functional studies

This comprehensive approach can help resolve discrepancies by identifying context-dependent effects or technical limitations in specific experimental settings .

How can AI-assisted approaches enhance WFIKKN antibody development and applications?

Artificial intelligence technologies are revolutionizing antibody development, including potential applications for WFIKKN research:

• RFdiffusion for antibody design: Recent advances in AI-driven protein design have enabled the generation of human-like antibodies with atomic precision. The Baker Lab's fine-tuned RFdiffusion model can design antibody loops—the intricate regions responsible for binding—creating functional antibodies computationally.

• Applications to WFIKKN research could include:

  • Designing antibodies with enhanced specificity to distinguish between WFIKKN1 and WFIKKN2

  • Creating antibodies that selectively block specific domain functions within WFIKKN proteins

  • Developing antibodies that detect specific conformational states during WFIKKN-growth factor interactions

• Implementation approaches:

  • Training models on existing WFIKKN structural data

  • Integrating binding site predictions with antibody design algorithms

  • Validating computationally designed antibodies against recombinant and native WFIKKN proteins

This computational approach could significantly accelerate WFIKKN research by producing highly specific research tools while reducing the time and resources required for traditional antibody development methods .

What is the therapeutic potential of WFIKKN-targeted approaches in muscle disorders?

Research on WFIKKN proteins, particularly their interaction with myostatin, suggests several promising therapeutic directions:

• Muscle wasting conditions: Overexpression of GASP-1/WFIKKN2 in mice produces a hypermuscular phenotype, suggesting therapeutic potential for:

  • Age-related sarcopenia

  • Muscular dystrophies

  • Cancer cachexia

  • Disuse atrophy

• Metabolic regulation: GASP-1 overexpression affects adiposity and glucose homeostasis, indicating potential applications in:

  • Type 2 diabetes

  • Metabolic syndrome

  • Obesity management

• Research methodologies to explore these applications include:

  • Development of domain-specific WFIKKN antibodies to modulate specific functions

  • Recombinant WFIKKN protein administration studies

  • Small molecule screening to identify compounds that enhance WFIKKN's myostatin inhibition

  • Targeted delivery systems to direct WFIKKN modulators to specific tissues

• Experimental models for testing:

  • Conditional transgenic animals

  • Patient-derived muscle cells

  • 3D muscle organoids

The dual role of WFIKKN proteins in regulating multiple growth factors makes them particularly interesting targets for developing nuanced therapeutic approaches that could modulate muscle growth while minimizing off-target effects .