SEMA4F Antibody, Biotin conjugated

What is SEMA4F protein and what are its primary biological functions?

SEMA4F (Semaphorin-4F) is a transmembrane class IV semaphorin family protein that plays critical roles in neural development. It is encoded by the SEMA4F gene (GeneID: 10505) and is also known by several synonyms including S4F, SEMAM, SEMAW, M-SEMA, and PRO2353 . The protein contains a sema domain, immunoglobulin domain (Ig), transmembrane domain (TM), and a short cytoplasmic domain .

Functionally, SEMA4F demonstrates growth cone collapse activity against retinal ganglion-cell axons, suggesting its importance in axonal guidance and neural patterning . Expression studies have detected SEMA4F postnatally in multiple tissues with highest levels in the brain, indicating its potential role in the maintenance of adult nervous tissue . Notably, SEMA4F serves as an important mediator of the association between normal Schwann cells and axons in the peripheral nervous system .

How is SEMA4F expressed during nervous system development and maintenance?

Quantitative RT-PCR analyses from sciatic nerves isolated at different developmental stages (postnatal days P0, P7, P14, P21, and P60) demonstrate that SEMA4F expression is detectable throughout postnatal development . Expression studies reveal that SEMA4F levels are as high in isolated Schwann cells as in whole nerve tissue, while being almost undetectable in fibroblasts, suggesting cell-type specific expression patterns .

In adult peripheral nerves where Schwann cell-axonal interactions are fully established, Schwann cells maintain significant SEMA4F expression. This expression pattern is not an artifact of tissue culture conditions but reflects the endogenous phenotype of Schwann cells in vivo . These observations highlight SEMA4F's potential importance in maintaining proper neural architecture in the adult peripheral nervous system.

What are the structural characteristics of SEMA4F Antibody, Biotin conjugated?

SEMA4F Antibody, Biotin conjugated is a polyclonal antibody typically raised in rabbit hosts that recognizes human SEMA4F protein . The antibody is generated against specific immunogens such as recombinant human Semaphorin-4F protein (amino acids 417-659) or fusion proteins containing the human sema domain, immunoglobulin domain, transmembrane domain, and short cytoplasmic domain .

The biotin conjugation process attaches biotin molecules to the antibody, enabling high-affinity interactions with streptavidin or avidin systems. This conjugation facilitates detection through enzyme-conjugated streptavidin in various assay formats . The molecular weight of the target protein (SEMA4F) is approximately 66 kDa, though it may also be observed at 84 kDa in some experimental settings, likely due to post-translational modifications .

What are the validated applications for SEMA4F Antibody, Biotin conjugated?

The primary validated application for SEMA4F Antibody, Biotin conjugated is ELISA (Enzyme-Linked Immunosorbent Assay) . When working with biotin-conjugated antibodies in general, they are commonly used in conjunction with streptavidin or avidin conjugates in various detection systems .

For non-biotin conjugated versions of SEMA4F antibody, additional validated applications include Western Blot (WB) and Immunoprecipitation (IP) . When designing experiments with the biotin-conjugated version, researchers should consider these potential applications while accounting for the additional considerations related to biotin-streptavidin detection systems.

Note: Optimal dilutions should be determined empirically for each experimental system .

How should I design appropriate controls when using SEMA4F Antibody, Biotin conjugated?

When designing experiments using SEMA4F Antibody, Biotin conjugated, include the following controls:

• Negative controls:

  • Isotype control: Use biotin-conjugated rabbit IgG (matching the host species and isotype) to assess non-specific binding

  • No primary antibody control: Replace primary antibody with buffer to detect potential non-specific binding of detection reagents

  • Blocking peptide control: Pre-incubate antibody with excess immunizing peptide to confirm specificity

• Positive controls:

  • Tissue/cell samples with known SEMA4F expression: Brain tissue or Schwann cells show reliable expression

  • Recombinant SEMA4F protein: Use as standard in ELISA applications

• Technical controls:

  • Streptavidin-only control: Apply only the streptavidin detection reagent to assess background

  • Endogenous biotin blocking: Apply avidin/biotin blocking steps when working with tissues containing endogenous biotin

These controls help distinguish genuine SEMA4F detection from technical artifacts and provide necessary validation for research findings .

What cell and tissue types are appropriate for studying SEMA4F expression?

Based on the literature, several cell and tissue types demonstrate reliable SEMA4F expression and are suitable for antibody validation and experimental applications:

• Neural tissues:

  • Brain tissue (highest expression levels)

  • Sciatic nerve (validated expression throughout development)

  • Retinal ganglion cells (functional target of SEMA4F activity)

• Cell lines and primary cells:

  • Schwann cells (high endogenous expression)

  • HepG2 cells (validated for IP applications with non-conjugated antibody)

  • Y79 cells (validated for WB applications with non-conjugated antibody)

• Negative or low expression controls:

  • Perineurial fibroblasts (almost undetectable expression)

  • Certain MPNST-derived tumor cell lines (NF90-8, ST88-14) show downregulation

When studying SEMA4F expression patterns, comparing levels across these different tissue types can provide valuable internal controls and contextual information for interpretation of results .

What are the optimal storage conditions for maintaining SEMA4F Antibody, Biotin conjugated activity?

To maintain optimal activity of SEMA4F Antibody, Biotin conjugated, adhere to these storage recommendations:

• Temperature: Store at -20°C or -80°C for long-term storage . For conjugated antibodies, avoid repeated freeze-thaw cycles by preparing small aliquots upon receipt.

• Short-term storage: Some biotin-conjugated antibodies may be stored at 4°C in the dark for up to 6 months , but this varies by manufacturer and should be verified for each specific product.

• Buffer conditions: Typical storage buffers contain stabilizers such as:

  • PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

  • 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride (pH 7.2)

  • Protein stabilizers such as BSA (10 mg/mL Bovine Serum Albumin)

• Light protection: Biotin conjugates should be protected from light exposure during storage and handling to prevent photobleaching and maintain conjugate integrity .

Proper storage is critical for maintaining antibody activity and experimental reproducibility over time.

How can I optimize signal-to-noise ratio in ELISA applications using SEMA4F Antibody, Biotin conjugated?

To optimize signal-to-noise ratio in ELISA applications:

• Antibody titration: Perform checkerboard titrations to determine optimal antibody concentration. Start with recommended dilutions (1:50 - 1:250 for most applications) and adjust based on signal intensity and background levels.

• Blocking optimization:

  • Use protein-free blocking buffers when possible to reduce background associated with biotin-containing proteins

  • Apply specialized avidin/biotin blocking steps to minimize endogenous biotin interference

  • Consider 1-2% BSA (immunoglobulin and protease-free) as a starting blocking agent

• Detection system considerations:

  • Select enzyme-conjugated streptavidin reagents compatible with desired detection methods

  • HRP-conjugated streptavidin typically provides good sensitivity with various substrates

  • Consider amplification systems (e.g., tyramide signal amplification) for detecting low-abundance targets

• Wash protocol optimization:

  • Increase number of wash steps (5-7 washes) to reduce non-specific binding

  • Include 0.05-0.1% Tween-20 in wash buffers to reduce hydrophobic interactions

  • Maintain consistent timing between wash steps for reproducible results

These optimization steps should be performed systematically, changing one variable at a time while maintaining appropriate controls .

What are the recommended protocols for antibody reconstitution and dilution?

For optimal reconstitution and dilution of SEMA4F Antibody, Biotin conjugated:

• Lyophilized antibody reconstitution:

  • Briefly centrifuge the vial before opening to collect material at the bottom

  • Reconstitute using deionized water or manufacturer-recommended buffer

  • Allow complete dissolution by gentle rotation or inversion (avoid vortexing)

  • For long-term storage, aliquot reconstituted antibody to minimize freeze-thaw cycles

• Working dilution preparation:

  • Use fresh, high-quality, and filter-sterilized buffers for dilutions

  • Recommended starting dilutions for biotin-conjugated antibodies:

    • ELISA applications: 1:50 - 1:1,000

    • Other applications: Determine empirically starting with 1:50 - 1:250

  • Prepare dilutions immediately before use for optimal performance

  • Include stabilizing proteins (0.1-0.5% BSA) in dilution buffers for dilute working solutions

• Buffer selection considerations:

  • Standard dilution buffer: PBS (pH 7.4) with 0.1% BSA

  • For reduced background: Include 0.05% Tween-20 in dilution buffer

  • For sensitive applications: Use specialized low cross-reactivity, biotin-free diluents

These protocols ensure maximal antibody activity and experimental reproducibility .

How does SEMA4F expression change in pathological conditions, and how can this be studied with SEMA4F Antibody, Biotin conjugated?

Research indicates significant alterations in SEMA4F expression in certain pathological conditions, particularly in neurological disorders and tumors. Studies have shown that SEMA4F is strongly downregulated in neurofibromas from NF1 patients compared to normal nerve tissue . Similarly, human MPNST-derived tumor cell lines show reduced SEMA4F expression compared to normal human Schwann cells .

To effectively study these expression changes:

• Comparative analysis approaches:

  • Quantitative ELISA using SEMA4F Antibody, Biotin conjugated to measure protein levels across normal and pathological samples

  • Tissue microarray analysis to evaluate expression patterns across multiple patient samples

  • Correlation of expression levels with clinical parameters and disease progression

• Experimental design considerations:

  • Include matched normal-pathological sample pairs whenever possible

  • Stratify samples by disease stage, grade, or genetic background

  • Normalize data to appropriate housekeeping proteins and include tissue-specific controls

• Data interpretation framework:

  • Establish baseline expression ranges in normal tissues

  • Quantify fold-changes relative to controls

  • Correlate protein expression with functional readouts (e.g., cell adhesion, migration)

These approaches can yield insights into the potential role of SEMA4F in disease pathogenesis and its utility as a biomarker .

How can I use SEMA4F Antibody, Biotin conjugated to study Schwann cell-axonal interactions?

SEMA4F plays a critical role in mediating Schwann cell-axonal interactions in the peripheral nervous system . To investigate these interactions using SEMA4F Antibody, Biotin conjugated:

• Co-culture experimental systems:

  • Establish Schwann cell-dorsal root ganglion (DRG) co-cultures

  • Apply biotin-conjugated SEMA4F antibody with fluorescent streptavidin detection

  • Use confocal microscopy to visualize SEMA4F localization at cell-cell interfaces

• Functional interaction studies:

  • Combine antibody labeling with live-cell imaging to track dynamic interactions

  • Perform antibody blocking experiments to assess functional consequences of SEMA4F inhibition

  • Correlate SEMA4F localization with cellular alignment and myelination patterns

• Analysis parameters:

  • Quantify co-localization coefficients between SEMA4F and axonal markers

  • Measure association and alignment indices between Schwann cells and axons

  • Assess changes in interaction dynamics following experimental manipulations

Studies have demonstrated that SEMA4F is localized to the Schwann cell plasma membrane and enriched at sites of axonal contact. Furthermore, SEMA4F expression is necessary for proper Schwann cell alignment with axons, as cells with downregulated SEMA4F show disrupted interactions . These experimental approaches can further elucidate the molecular mechanisms underlying these critical neural interactions.

What are the technical challenges in detecting SEMA4F in complex neural tissues, and how can they be overcome?

Detecting SEMA4F in complex neural tissues presents several technical challenges:

• Tissue complexity challenges:

  • High cellular heterogeneity in neural tissues

  • Presence of myelin and lipid-rich structures that can increase background

  • Potential masking of epitopes within dense cellular networks

• SEMA4F-specific considerations:

  • Transmembrane localization may require membrane permeabilization optimization

  • Expression levels vary by developmental stage and cell type

  • Multiple isoforms (observed at 66 kDa and 84 kDa)

• Biotin-conjugate specific issues:

  • Endogenous biotin in neural tissues can increase background

  • Potential cross-reactivity with biotin-containing proteins

  • Signal amplification challenges in low-expression regions

Recommended solutions:

• Tissue preparation optimization:

  • Test multiple fixation protocols (4% PFA, methanol, acetone) to identify optimal epitope preservation

  • Employ antigen retrieval methods (citrate buffer, pH 6.0)

  • Use thinner tissue sections (5-10 μm) for better antibody penetration

• Signal enhancement strategies:

  • Implement tyramide signal amplification (TSA) for low abundance detection

  • Use high-sensitivity streptavidin-conjugated detection systems

  • Employ sequential multiplexing approaches for co-localization studies

• Background reduction techniques:

  • Apply stringent avidin/biotin blocking steps before antibody incubation

  • Include detergents (0.1-0.3% Triton X-100) in blocking buffers

  • Extend washing steps (6-8 washes) with gentle agitation

These approaches can substantially improve the signal-to-noise ratio when detecting SEMA4F in complex neural tissues, enabling more precise localization and quantification studies .

How should I interpret variations in SEMA4F molecular weight observed in different experimental systems?

The SEMA4F protein has a calculated molecular weight of approximately 66 kDa based on its 615 amino acid sequence, but it is also frequently observed at 84 kDa in certain experimental systems . These variations require careful interpretation:

• Potential causes of molecular weight variations:

  • Post-translational modifications (glycosylation, phosphorylation)

  • Tissue-specific processing or alternative splicing

  • Species-specific differences in protein modification

  • Experimental conditions affecting protein migration

• Validation approaches:

  • Compare observed bands with recombinant protein standards

  • Perform peptide competition assays to confirm specificity of each band

  • Analyze samples under reducing and non-reducing conditions

  • Employ deglycosylation enzymes to assess contribution of glycosylation

• Interpretation framework:

  • Document consistent patterns across experimental replicates

  • Compare observations with published literature

  • Consider tissue-specific or cell-type-specific expression patterns

  • Evaluate correlation between band intensity and functional outcomes

The molecular weight variations observed with SEMA4F antibodies likely reflect biologically relevant modifications of the protein rather than non-specific binding, as these patterns have been consistently observed across multiple studies and antibody preparations .

What methods can I use to validate the specificity of SEMA4F Antibody, Biotin conjugated in my experimental system?

To rigorously validate the specificity of SEMA4F Antibody, Biotin conjugated:

• Molecular validation approaches:

  • Peptide competition/blocking: Pre-incubate antibody with excess immunizing peptide

  • RNA interference: Compare detection in SEMA4F-knockdown versus control samples

  • Genetic knockout models: Test antibody in SEMA4F-null tissues/cells when available

  • Heterologous expression: Test detection in SEMA4F-transfected versus non-transfected cells

• Technical validation strategies:

  • Cross-platform validation: Compare results between ELISA, Western blot, and immunostaining

  • Multiple antibody validation: Compare results with independent SEMA4F antibodies targeting different epitopes

  • Species cross-reactivity assessment: Test antibody performance across relevant species models

• Experimental design considerations:

  • Include both positive controls (tissues known to express SEMA4F, e.g., brain)

  • Include negative controls (tissues with minimal expression, e.g., fibroblasts)

  • Employ tissue-specific internal controls (non-target proteins) to verify assay performance

The combination of these approaches provides comprehensive validation of antibody specificity, ensuring reliable interpretation of experimental results .

How can I quantitatively analyze SEMA4F expression data from ELISA applications for comparative studies?

For rigorous quantitative analysis of SEMA4F expression data from ELISA applications:

• Standard curve optimization:

  • Use recombinant SEMA4F protein as standard

  • Prepare standards in the same matrix as experimental samples

  • Employ a minimum of 7-8 concentration points in duplicate or triplicate

  • Use four-parameter logistic regression for curve fitting

• Data normalization strategies:

  • Normalize to total protein concentration for tissue/cell lysates

  • Use housekeeping proteins as internal references when appropriate

  • Consider sample-specific normalization factors for different tissue types

• Statistical analysis framework:

  • Calculate coefficient of variation (CV) for technical replicates (<15% acceptable)

  • Determine limits of detection (LOD) and quantification (LOQ)

  • Apply appropriate statistical tests based on data distribution

  • Consider paired tests for matched normal/pathological samples

• Comparative analysis approach:

  • Express results as fold-change relative to appropriate controls

  • Construct hierarchical clustering for pattern identification across sample groups

  • Correlate SEMA4F levels with other molecular or clinical parameters

  • Perform time-course analyses for developmental or intervention studies

This systematic approach to quantitative analysis enhances the reproducibility and interpretability of SEMA4F expression data across experimental conditions and sample types .

What are the common technical issues encountered when using SEMA4F Antibody, Biotin conjugated, and how can they be resolved?

Researchers commonly encounter several technical challenges when working with SEMA4F Antibody, Biotin conjugated:

• High background signal:

  • Possible causes: Endogenous biotin in samples, insufficient blocking, non-specific binding

  • Solutions:

    • Implement avidin/biotin blocking steps before antibody incubation

    • Use biotin-free blocking reagents

    • Increase washing steps (frequency and duration)

    • Optimize antibody dilution through titration experiments

• Weak or absent signal:

  • Possible causes: Low SEMA4F expression, epitope masking, antibody degradation

  • Solutions:

    • Verify SEMA4F expression in your sample type with literature

    • Test multiple antigen retrieval methods

    • Check antibody storage conditions and date of receipt

    • Consider signal amplification systems (e.g., TSA)

• Inconsistent results between replicates:

  • Possible causes: Pipetting errors, inconsistent tissue processing, antibody instability

  • Solutions:

    • Standardize all protocol steps with precise timing

    • Prepare master mixes to reduce pipetting variations

    • Process all comparative samples simultaneously

    • Aliquot antibody to avoid repeated freeze-thaw cycles

• Cross-reactivity concerns:

  • Possible causes: Antibody binding to related semaphorin family proteins

  • Solutions:

    • Perform validation in SEMA4F-deficient control samples

    • Compare results with alternative SEMA4F antibodies

    • Include peptide competition controls

Systematic troubleshooting of these common issues can significantly improve experimental outcomes when working with SEMA4F Antibody, Biotin conjugated .

How should I address potential interference from endogenous biotin when using SEMA4F Antibody, Biotin conjugated?

Endogenous biotin can significantly interfere with detection systems using biotin-conjugated antibodies, particularly in biotin-rich tissues such as brain, kidney, and liver:

• Prevention strategies:

  • Avidin/biotin blocking: Apply unconjugated avidin followed by excess biotin before antibody incubation

  • Commercial blocking kits: Use specialized endogenous biotin blocking kits designed for immunoassays

  • Sample pretreatment: Consider mild oxidation of endogenous biotin in fixed tissues (0.01% hydrogen peroxide)

• Alternative detection approaches:

  • Two-step detection: Use unconjugated primary SEMA4F antibody followed by biotinylated secondary and streptavidin

  • Non-biotin detection systems: Consider directly conjugated fluorescent antibodies for critical applications

  • Polymer-based detection: Employ polymer-conjugated detection systems that avoid biotin-streptavidin interaction

• Experimental design considerations:

  • Tissue-specific controls: Include no-primary-antibody controls for each tissue type

  • Endogenous biotin mapping: Perform streptavidin-only controls to identify high-biotin regions

  • Cross-validation: Confirm key findings with non-biotin detection methods

These approaches can effectively mitigate the impact of endogenous biotin on experimental results, ensuring more reliable detection of SEMA4F protein .

How is SEMA4F Antibody being used to investigate neurological disorders and tumor biology?

Recent research has begun to elucidate the potential role of SEMA4F in neurological disorders and tumor biology:

• Neurofibromatosis Type 1 (NF1) research:

  • SEMA4F is strongly downregulated in neurofibromas from NF1 patients compared to normal nerve tissue

  • SEMA4F Antibody is being used to investigate the molecular mechanisms underlying disrupted Schwann cell-axonal interactions in NF1

  • Comparative studies of SEMA4F expression in different NF1-associated tumor types provide insights into disease progression

• Cancer biology applications:

  • SEMA4F is downregulated in human MPNST-derived tumor cell lines compared to normal Schwann cells

  • Researchers are investigating whether SEMA4F could serve as a potential biomarker for certain neural tumors

  • Studies are examining correlations between SEMA4F expression levels and tumor invasiveness or metastatic potential

• Neurodevelopmental disorder research:

  • Given SEMA4F's role in neural development, researchers are exploring its potential involvement in neurodevelopmental disorders

  • SEMA4F Antibody is being used to map expression patterns across developmental timepoints in normal and disease models

  • Investigation of SEMA4F interactions with other guidance molecules may reveal novel therapeutic targets

These emerging applications highlight the potential of SEMA4F Antibody as a valuable tool for understanding the molecular basis of neurological disorders and tumor biology .

What are the considerations for using SEMA4F Antibody, Biotin conjugated in multiplex detection systems?

When incorporating SEMA4F Antibody, Biotin conjugated into multiplex detection systems:

• Compatibility considerations:

  • Spectral overlap: Select fluorophore-conjugated streptavidins with minimal spectral overlap with other detection channels

  • Cross-reactivity: Test for potential cross-reactivity between multiple primary antibodies

  • Signal balance: Optimize dilutions to achieve balanced signal intensity across all targets

• Sequential multiplex approaches:

  • Stripping and reprobing: Consider gentle elution buffers to remove antibodies between rounds

  • Permanent labeling: Use methods like tyramide signal amplification for sequential rounds

  • Multi-round imaging: Implement image registration algorithms for accurate co-localization analysis

• Advanced multiplexing technologies:

  • Mass cytometry: Consider metal-tagged streptavidin for mass cytometry applications

  • Spectral imaging: Utilize spectral unmixing algorithms to separate overlapping signals

  • Proximity ligation: Combine with secondary proximity probes for detecting protein-protein interactions

• Control and validation strategies:

  • Single-color controls: Prepare samples with each antibody alone to establish spectral profiles

  • Biological controls: Include samples with known co-expression or mutually exclusive expression patterns

  • Spillover matrices: Calculate and apply compensation matrices for fluorescent signals

These considerations enable robust multiplex detection of SEMA4F alongside other proteins of interest in complex biological samples .

What new applications of SEMA4F Antibody, Biotin conjugated might emerge in neurodevelopmental research?

Several promising future applications of SEMA4F Antibody, Biotin conjugated in neurodevelopmental research include:

• High-resolution spatial mapping:

  • Super-resolution microscopy to map SEMA4F distribution at the nanoscale within growth cones and axonal guidance structures

  • Spatial transcriptomics combined with protein detection to correlate SEMA4F protein localization with local gene expression patterns

  • 3D tissue clearing and whole-mount imaging to visualize SEMA4F distribution across intact neural circuits

• Developmental dynamics investigations:

  • In vivo imaging using biotinylated antibody fragments to track SEMA4F expression in developing systems

  • Correlation of SEMA4F localization with dynamic cellular behaviors during critical developmental windows

  • Investigation of SEMA4F's role in activity-dependent neural circuit refinement and plasticity

• Pathological model applications:

  • Comparative analysis of SEMA4F expression and localization in neurodevelopmental disorder models

  • Investigation of SEMA4F as a potential biomarker for early detection of neural pathologies

  • Assessment of therapeutic interventions targeting SEMA4F-mediated developmental pathways

These emerging applications could significantly advance our understanding of SEMA4F's role in neural development and related disorders .

How might technical advances improve the utility of SEMA4F Antibody, Biotin conjugated in research applications?

Future technical advances likely to enhance the utility of SEMA4F Antibody, Biotin conjugated include:

• Next-generation conjugation technologies:

  • Site-specific biotin conjugation to preserve antigen-binding capacity

  • Controllable biotin-to-antibody ratios for optimized detection sensitivity

  • Cleavable linker technologies for signal amplification with reduced background

• Advanced detection platforms:

  • Single-molecule detection systems for quantifying low-abundance SEMA4F expression

  • Microfluidic-based detection platforms for automated, high-throughput analysis

  • Label-free detection systems utilizing surface plasmon resonance with biotinylated capture antibodies

• Integration with emerging omics approaches:

  • Spatial proteomics combining SEMA4F antibody detection with mass spectrometry

  • Single-cell proteomics workflows incorporating SEMA4F detection

  • Multi-omic data integration frameworks correlating SEMA4F protein levels with transcriptomic and metabolomic profiles

• AI-enhanced image analysis:

  • Deep learning algorithms for automated identification of SEMA4F-positive structures

  • Pattern recognition approaches for characterizing SEMA4F distribution in complex tissues

  • Predictive modeling of SEMA4F expression changes based on experimental interventions

These technological advances promise to expand the utility of SEMA4F Antibody, Biotin conjugated across diverse research applications, from basic neurodevelopmental studies to complex disease investigations .