FBN2 Antibody, HRP conjugated

What is FBN2 and why is it an important research target?

FBN2 (Fibrillin-2) belongs to the fibrillin family of proteins that polymerize into microfibrils, providing structural support for both elastic and non-elastic connective tissues. In humans, the canonical protein has a length of 2912 amino acid residues and a mass of 314.8 kDa . It is primarily expressed in the placenta and is critically involved in eye development and carbohydrate metabolism pathways . FBN2 is of particular research interest because mutations in the FBN2 gene cause Congenital Contractural Arachnodactyly (CCA), a rare autosomal dominant connective tissue disorder characterized by crumpled ears, arachnodactyly, camptodactyly, and thoracolumbar scoliosis . The protein undergoes significant post-translational modifications, including O-glycosylation and N-glycosylation, which can affect its function and detection .

What are the standard applications for HRP-conjugated FBN2 antibodies?

HRP-conjugated FBN2 antibodies are valuable research tools primarily used in the following applications:

HRP conjugation provides enzymatic signal amplification, resulting in enhanced sensitivity when paired with appropriate substrates. For FBN2 detection, these antibodies have been reported to work effectively across multiple species including human and mouse samples . The method choice depends on whether you're investigating expression levels, localization, or protein interactions.

How should FBN2 antibody specificity be validated prior to experimental use?

Validating FBN2 antibody specificity is critical to ensure reliable research outcomes. A comprehensive validation protocol should include:

• Western blot analysis using positive and negative controls: Look for a single band at approximately 315 kDa in tissues known to express FBN2 (e.g., placenta). Absence of bands in tissues with minimal FBN2 expression serves as negative control.

• Genetic knockout/knockdown validation: Compare antibody reactivity in wild-type versus FBN2 knockout/knockdown samples. Research on FBN2 has employed fbn2 mutant plants as negative controls to identify non-specific binding .

• Cross-reactivity testing: Confirm the antibody does not recognize related proteins like FBN1a or FBN1b. This has been demonstrated through immunoblotting under both native and denaturing conditions .

• Peptide competition assay: Pre-incubation of the antibody with excess FBN2 peptide should abolish specific staining.

• Multiple antibody concordance: Utilize multiple antibodies targeting different epitopes of FBN2 to verify consistent detection patterns.

Validation ensures that experimental observations reflect true FBN2 biology rather than artifacts of non-specific antibody binding.

What sample preparation methods optimize FBN2 detection with HRP-conjugated antibodies?

Effective sample preparation is crucial for reliable FBN2 detection due to its large size and extensive post-translational modifications. Optimize your protocol with these methodological considerations:

For protein extraction:

• Use buffer systems containing 1% Triton X-100, which has been demonstrated to efficiently solubilize FBN2 from membrane-associated structures like plastoglobules in plant systems .

• Include protease inhibitors to prevent degradation of this large protein.

• Consider gentle lysis methods that preserve protein complexes if studying FBN2 interactions.

For Western blotting:

• Use gradient gels (4-12% or 3-8%) to effectively resolve this large 315 kDa protein.

• Extend transfer time (overnight) at lower voltage with SDS-containing transfer buffer to facilitate complete transfer of large proteins.

• Include a protein molecular weight ladder that extends to at least 350 kDa.

For immunoprecipitation studies:

• Sequential extraction approaches may be necessary, as demonstrated in studies where FBN2 exhibits dual localization (soluble and membrane-associated fractions) .

• When working with membrane-associated FBN2, solubilization with 0.01% Triton X-100 prior to immunoprecipitation has proven effective .

These methodological refinements significantly improve detection sensitivity and specificity when working with this challenging protein target.

How can researchers distinguish between FBN2 isoforms using HRP-conjugated antibodies?

Distinguishing between FBN2 isoforms requires careful antibody selection and experimental design:

• Epitope mapping: Determine whether your HRP-conjugated antibody recognizes an epitope common to all isoforms or is isoform-specific. Request epitope information from manufacturers or perform epitope mapping experiments.

• Electrophoretic resolution: Up to 2 different isoforms have been reported for FBN2 . Design gel systems that can resolve potential small differences in molecular weight between isoforms.

• Isoform-specific controls: Generate positive controls expressing specific isoforms through recombinant expression systems.

• Complementary techniques:

  • Use RT-PCR with isoform-specific primers to correlate protein detection with transcript expression.

  • Consider mass spectrometry following immunoprecipitation to identify isoform-specific peptides.

• Domain-specific antibodies: For research questions requiring isoform distinction, consider using multiple domain-specific antibodies, as mutations in FBN2 are predominantly found in the central stretch of calcium-binding epidermal growth factor-like (cbEGF-like) domains (exons 24-35) .

This multi-faceted approach allows researchers to confidently identify specific FBN2 isoforms in their experimental systems.

How should researchers address non-specific background when using HRP-conjugated FBN2 antibodies?

High background is a common challenge when working with HRP-conjugated antibodies for FBN2 detection. Implement these evidence-based solutions:

• Optimize blocking conditions: Test different blocking agents (BSA, milk, commercial blockers) at various concentrations (3-5%) and times (1-2 hours). For FBN2 detection, BSA-based blockers may be preferable as milk contains glycoproteins that might cross-react.

• Adjust antibody concentration: Titrate the HRP-conjugated FBN2 antibody to determine the optimal concentration that maximizes specific signal while minimizing background. Based on published methodologies, initial dilutions of 1:1000-1:5000 for Western blots are recommended .

• Implement additional washing steps: Increase the number and duration of wash steps using TBS-T or PBS-T (0.05-0.1% Tween-20).

• Use specific controls: Include fbn2 knockout/mutant samples as negative controls to identify non-specific binding, as demonstrated in plant-based FBN2 research .

• Consider signal enhancement systems: For weak signals, enhance detection using chemiluminescent substrates with varying sensitivity rather than increasing antibody concentration, which can increase background.

• Pre-adsorption: Pre-adsorb the antibody with tissue/cell lysate from organisms or tissues that do not express FBN2 to remove antibodies that bind to conserved epitopes.

Implementation of these methodological refinements can significantly improve signal-to-noise ratio in FBN2 detection experiments.

What approaches can resolve contradictory FBN2 localization data obtained using HRP-conjugated antibodies?

Contradictory FBN2 localization data is not uncommon due to the protein's complex distribution patterns. Research shows that FBN2 can exhibit dual localization, as observed in plant systems where it is found in both stromal and membrane-associated fractions . To resolve conflicting localization data:

• Employ subcellular fractionation: Systematically separate cellular compartments and analyze FBN2 distribution across fractions. In plants, FBN2 has been successfully separated into soluble and membrane fractions, revealing distinct functions based on localization .

• Validate with orthogonal methods:

  • Complement immunodetection with GFP-tagged FBN2 expression

  • Use proximity ligation assays to confirm interaction partners in specific compartments

  • Apply super-resolution microscopy techniques for precise localization

• Control for fixation artifacts: Different fixation methods can alter epitope accessibility and apparent protein localization. Compare results using multiple fixation protocols (e.g., paraformaldehyde, methanol, acetone).

• Consider developmental and physiological states: FBN2 expression and localization may change during development or under stress conditions. In plants, FBN2's protective role against abiotic stresses suggests its localization might be dynamic .

• Analyze context-dependent interactions: Co-immunoprecipitation studies revealed that FBN2 interacts with different proteins depending on its localization. For example, in plant systems, soluble FBN2 interacts with fructose bisphosphate aldolase, while membrane-associated FBN2 interacts with a distinct set of proteins .

This systematic approach can resolve apparently contradictory data by revealing the dynamic and context-dependent nature of FBN2 localization.

How can HRP-conjugated FBN2 antibodies facilitate research on Congenital Contractural Arachnodactyly (CCA)?

HRP-conjugated FBN2 antibodies serve as valuable tools for investigating the molecular pathology of Congenital Contractural Arachnodactyly (CCA). This rare autosomal dominant disorder is caused by mutations in the FBN2 gene, with pathogenic variants primarily concentrated in exons 24-35 . Research applications include:

• Mutant protein expression analysis: HRP-conjugated antibodies can detect altered expression levels of FBN2 in patient-derived samples. This is particularly relevant for novel mutations such as the recently identified c.3472G>C (p.Asp1158His) in exon 26 .

• Structural impact assessment: Immunodetection can reveal whether specific mutations affect protein stability or cellular localization. Recent research demonstrated that the p.Asp1158His mutation changes amino acid properties from acidic to basic, disrupting hydrogen bonding with Asn1176 .

• Genotype-phenotype correlation studies: Combine immunodetection of FBN2 with clinical phenotyping to establish correlations between specific mutations and disease severity or presentation.

• Therapeutic screening: Antibody-based assays can evaluate potential therapeutics that might restore normal FBN2 function or expression.

• Diagnostic development: Research using these antibodies contributes to improved diagnostic approaches, as indicated by recent findings that "provide new insights for the diagnosis of CCA and may have an impact on genetic counseling" .

When studying CCA-associated mutations, researchers should consider domain-specific antibodies, as most pathogenic mutations occur in the calcium-binding EGF-like domains encoded by exons 24-35 .

What methodological considerations apply when studying FBN2 protein-protein interactions using HRP-conjugated antibodies?

Investigating FBN2 protein-protein interactions requires specialized approaches due to the protein's large size, complex structure, and tendency to form high-molecular-weight complexes. Based on successful interaction studies, consider these methodological recommendations:

• Co-immunoprecipitation optimization:

  • Use specific polyclonal antibodies against full-length FBN2 for immunoprecipitation

  • For membrane-associated FBN2, solubilize with 0.01% Triton X-100 prior to immunoprecipitation

  • Perform parallel analysis with FBN2-knockout/mutant samples to identify non-specific interactions

  • Multiple replication is essential - successful studies typically perform at least three independent Co-IP experiments

• Mass spectrometry analysis:

  • Use high-resolution mass spectrometry to identify co-precipitated proteins

  • Filter against proteins found in control immunoprecipitations using FBN2-deficient samples

  • Consider removing highly abundant proteins that may represent non-specific binding

• Validation of identified interactions:

  • Confirm interactions using reverse co-immunoprecipitation

  • Employ proximity ligation assays for in situ validation

  • Use FRET/BRET approaches for dynamic interaction studies

• Domain mapping:

  • Design experiments to identify which domains of FBN2 mediate specific interactions

  • Consider the calcium-binding EGF-like domains, which are frequently affected in disease-causing mutations

This approach has successfully identified FBN2 interaction partners in different systems, revealing important functional relationships. For example, in plants, FBN2 was found to interact with APE1, explaining its role in protecting photosystem II against abiotic stresses .

How can researchers effectively measure alterations in FBN2 expression across different experimental conditions?

Quantifying changes in FBN2 expression requires careful methodological considerations due to its large size and varying expression levels across tissues. Implement these evidence-based approaches:

• Western blot quantification:

  • Use HRP-conjugated FBN2 antibodies with gradient gels (3-8% or 4-12%) to efficiently resolve this 315 kDa protein

  • Include appropriate loading controls (avoid using housekeeping proteins affected by your experimental conditions)

  • Employ digital image acquisition and analysis software for accurate densitometry

  • Present data as fold-change relative to control conditions with appropriate statistical analysis

• ELISA-based quantification:

  • Develop sandwich ELISA using capture and HRP-conjugated detection antibodies against different FBN2 epitopes

  • Generate standard curves using recombinant FBN2 protein for absolute quantification

  • Validate assay linearity, sensitivity, and specificity for your experimental system

• qRT-PCR correlation:

  • Complement protein detection with transcript analysis

  • Design primers spanning exon-exon junctions, particularly focusing on the central region (exons 24-35) where pathogenic mutations frequently occur

  • Normalize to multiple reference genes selected for stability under your experimental conditions

• Experimental controls:

  • Include positive controls (tissues with known high FBN2 expression, like placenta)

  • Use FBN2-deficient samples as negative controls when available

  • Consider the impact of experimental conditions on post-translational modifications, which may affect antibody recognition

This multi-method approach provides robust quantification of FBN2 expression changes, essential for understanding its role in development, disease mechanisms, and stress responses.

How can AlphaFold2 and other structural prediction tools complement FBN2 antibody-based research?

Integrating structural prediction tools with antibody-based detection creates powerful research synergies for FBN2 investigations:

• Epitope accessibility prediction:

  • Use AlphaFold2-predicted structures to assess epitope accessibility in native FBN2 protein

  • Select antibodies targeting exposed regions for improved detection sensitivity

  • Recent research has successfully employed AlphaFold2 to predict structural changes in FBN2 caused by the p.Asp1158His mutation

• Mutation impact assessment:

  • Predict structural changes caused by disease-associated mutations

  • Target antibodies to regions predicted to undergo conformational changes

  • Correlate structural predictions with experimental antibody binding patterns

• Domain-specific investigations:

  • Design research strategies focusing on the calcium-binding EGF-like domains (exons 24-35), where most pathogenic mutations occur

  • Use domain-specific antibodies to validate structural predictions about domain interactions

• Protein-protein interaction surfaces:

  • Identify potential interaction surfaces from structural predictions

  • Design co-immunoprecipitation experiments targeting predicted interaction domains

  • Validate in silico predictions with antibody-based interaction studies

This integrated approach combines computational prediction with experimental validation, providing deeper insights into FBN2 structure-function relationships and disease mechanisms.

What considerations apply when using FBN2 antibodies in single-cell protein analysis technologies?

Emerging single-cell protein analysis technologies offer unprecedented insights into FBN2 expression heterogeneity, but require specific methodological considerations:

• Single-cell Western blotting:

  • Optimize cell lysis conditions to efficiently solubilize FBN2 from both soluble and membrane-associated pools

  • Employ microfluidic platforms capable of resolving high molecular weight proteins

  • Use highly specific HRP-conjugated FBN2 antibodies at optimized concentrations

• Mass cytometry (CyTOF):

  • Metal-conjugated FBN2 antibodies must be validated for specificity

  • Include antibodies against known FBN2 interaction partners identified in co-immunoprecipitation studies

  • Develop analysis pipelines that can correlate FBN2 levels with cellular phenotypes

• Microfluidic immunofluorescence:

  • Optimize fixation and permeabilization protocols to maintain cellular architecture while allowing antibody access to FBN2

  • Consider the dual localization of FBN2 observed in some systems when interpreting subcellular distribution patterns

• Proximity ligation assays at single-cell resolution:

  • Use FBN2 antibodies in combination with antibodies against interacting partners

  • Verify specificity using FBN2-deficient cells as negative controls

  • Quantify interaction signals in the context of cellular heterogeneity

These advanced approaches reveal cell-to-cell variability in FBN2 expression, localization, and interaction patterns, providing insights into its functional roles in development and disease that are not apparent in bulk analysis.