SEMA6D Antibody, HRP conjugated

What is SEMA6D and what are its key structural features?

SEMA6D (Semaphorin-6D) is a 1073 amino acid transmembrane protein belonging to the semaphorin family. Its structure features one PSI domain and one semaphorin domain, which are crucial for mediating interactions with other proteins and signaling pathways that influence neuronal growth and guidance. SEMA6D is primarily localized to the cell membrane or cytoplasm, depending on the specific isoform . The protein undergoes alternative splicing, resulting in seven distinct isoforms that may serve different functional roles in neural development and plasticity .

What is the primary biological function of SEMA6D in neural development?

SEMA6D plays a critical role in the development and maintenance of neuronal connections. It functions primarily by providing a stop signal for dorsal root ganglion neurons as they reach their target areas, thereby guiding the intricate wiring of the nervous system . This signaling mechanism is essential for the remodeling of neuronal networks during development and potentially in plasticity throughout the lifespan .

How does SEMA6D expression vary across different tissues?

According to Human Protein Atlas analysis, SEMA6D expression is relatively higher in kidney tissue compared to other normal tissues . This tissue-specific expression pattern suggests potentially specialized functions in different organ systems, which may explain its varied roles in different pathological conditions, particularly in various cancer types .

What are the key differences between polyclonal and monoclonal SEMA6D antibodies?

Polyclonal SEMA6D antibodies, such as the rabbit-derived product from AFG Scientific, are generated from multiple B cell lineages and recognize different epitopes on the SEMA6D protein . These antibodies offer broader antigen recognition but may have batch-to-batch variability.

In contrast, monoclonal antibodies like the mouse-derived A-8 clone from Santa Cruz Biotechnology are produced from a single B cell clone, recognizing a single epitope. The SEMA6D Antibody (A-8) is a mouse monoclonal IgG2a kappa light chain antibody that detects SEMA6D protein from mouse, rat, and human origins . Monoclonal antibodies provide higher specificity and consistency between batches, making them valuable for applications requiring precise epitope targeting.

What experimental applications are SEMA6D antibodies suitable for?

SEMA6D antibodies have validated applications across multiple experimental techniques:

For experiments requiring detection of multiple epitopes, polyclonal antibodies may offer better sensitivity, while monoclonal antibodies are preferable for applications demanding high specificity and reproducibility across experiments .

What are the optimal storage conditions for maintaining SEMA6D antibody activity?

For maintaining maximum activity of SEMA6D antibodies, store at -20°C or -80°C upon receipt . Avoid repeated freeze-thaw cycles, as these can degrade antibody quality and reduce binding efficacy . For HRP-conjugated antibodies, storing in buffer containing glycerol (such as the 50% glycerol, 0.01M PBS, pH 7.4 formulation) helps maintain stability during freeze-thaw transitions . When working with the antibody, aliquoting into single-use volumes is recommended to prevent repeated freezing and thawing of the stock solution.

How should researchers optimize ELISA protocols using HRP-conjugated SEMA6D antibodies?

When optimizing ELISA protocols with HRP-conjugated SEMA6D antibodies, consider these methodological steps:

• Antibody titration: Perform a dilution series (typically 1:500 to 1:5000) to determine optimal antibody concentration that maximizes signal-to-noise ratio.

• Blocking optimization: Use 1-5% BSA or non-fat milk in PBS or TBS to minimize background signal.

• Incubation conditions: Test various incubation times (1-4 hours) and temperatures (room temperature vs. 4°C) to enhance specific binding while minimizing non-specific interactions.

• Substrate selection: Choose appropriate HRP substrates based on desired sensitivity and detection method (TMB for colorimetric detection, luminol-based reagents for chemiluminescence).

• Preservation considerations: The buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 helps maintain antibody stability during the assay .

What methodological considerations are important when using SEMA6D antibodies for Western blotting?

For optimal Western blotting results with SEMA6D antibodies, researchers should consider:

• Sample preparation: Complete cell lysis is critical for membrane-associated proteins like SEMA6D. Consider using detergent mixtures (RIPA or NP-40 based) with protease inhibitors.

• Protein denaturation: Given SEMA6D's complex structure with PSI and semaphorin domains, optimize denaturation conditions (temperature, reducing agents) to ensure epitope exposure while preventing aggregation.

• Transfer optimization: For the 1073 amino acid SEMA6D protein, use lower percentage gels (6-8%) and extended transfer times for complete transfer of high molecular weight proteins.

• Blocking and antibody dilution: For HRP-conjugated antibodies, optimize blocking (typically 3-5% BSA or milk) to minimize background without interfering with specific binding .

• Detection system selection: For HRP-conjugated antibodies, choose enhanced chemiluminescence (ECL) substrates with sensitivity appropriate for your expected protein levels .

How is SEMA6D expression altered in clear cell renal cell carcinoma (ccRCC)?

SEMA6D expression is markedly lower in ccRCC tissues compared to adjacent normal tissues . This downregulation has been validated through multiple methodologies including RNA-sequencing data analysis from the TCGA-KIRC database, qRT-PCR verification, immunohistochemistry (IHC), and ELISA testing of urine samples from ccRCC patients compared to healthy volunteers . The area under the ROC curve for SEMA6D as a diagnostic marker was 0.791 with a cutoff value of 265.1 ng/L, suggesting potential utility as a diagnostic biomarker .

What is the prognostic significance of SEMA6D expression in cancer research?

SEMA6D expression has significant prognostic implications that vary by cancer type:

These contrasting roles across different cancer types highlight the context-dependent function of SEMA6D in tumor biology and the importance of tissue-specific research approaches.

What experimental approaches can be used to investigate SEMA6D as a potential biomarker?

When investigating SEMA6D as a potential biomarker, researchers should consider these methodological approaches:

• Expression analysis: Utilize qRT-PCR, Western blotting with HRP-conjugated antibodies, and immunohistochemistry to quantify SEMA6D expression in clinical samples compared to controls .

• Bioinformatic validation: Incorporate database analysis using platforms like GEPIA, TCGA-KIRC, and Human Protein Atlas to validate expression patterns across larger patient cohorts .

• Liquid biopsy development: Consider ELISA-based detection in urine samples, which has shown promise for ccRCC with an area under the ROC curve of 0.791 .

• Correlation with clinical features: Analyze relationships between SEMA6D expression and clinical parameters including tumor stage, grade, and patient survival through Kaplan-Meier survival analysis and hazard ratio calculations .

• Functional validation: Employ gain and loss of function experiments in cell lines to determine the biological effects of SEMA6D alteration on cancer cell phenotypes .

How can researchers effectively distinguish between the seven alternative splice isoforms of SEMA6D?

To effectively distinguish between the seven alternative splice isoforms of SEMA6D:

• Isoform-specific PCR primers: Design primers spanning exon-exon junctions unique to each isoform.

• Western blotting optimization: Use gradient gels (4-12%) to separate isoforms with subtle size differences, coupled with HRP-conjugated antibodies for detection .

• Immunoprecipitation approach: Perform IP with monoclonal antibodies followed by mass spectrometry for precise isoform identification .

• Domain-specific antibodies: Consider using antibodies targeting regions present or absent in specific isoforms.

• Subcellular localization analysis: Utilize immunofluorescence with confocal microscopy to distinguish between membrane-localized and cytoplasmic isoforms .

Understanding isoform-specific functions of SEMA6D is critical as these may have distinct roles in neural development and pathological conditions.

What methods can help troubleshoot non-specific binding when using SEMA6D antibodies?

When encountering non-specific binding with SEMA6D antibodies, consider these methodological solutions:

• Antibody validation: Verify antibody specificity using positive and negative control tissues or cell lines with known SEMA6D expression levels.

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, normal serum) and concentrations to reduce background without compromising specific signal.

• Antibody titration: Perform careful dilution series to identify the optimal concentration that maximizes specific binding while minimizing background.

• Buffer modifications: Adjust salt concentration (150-500 mM NaCl) and detergent levels (0.05-0.3% Tween-20) in wash buffers to reduce non-specific interactions.

• Pre-adsorption controls: For polyclonal antibodies, consider pre-adsorbing with recombinant antigen or using peptide-blocking experiments to confirm specificity .

• Consider alternative detection systems: If HRP-conjugated antibodies show high background, test unconjugated primary with separate HRP-conjugated secondary antibodies for comparison.

How can SEMA6D antibodies be used to investigate its interactions with plexin and other binding partners?

To investigate SEMA6D interactions with plexins and other binding partners:

• Co-immunoprecipitation: Use SEMA6D antibodies for IP followed by Western blotting for suspected binding partners. HRP-conjugated SEMA6D antibodies can be used for direct detection in reverse co-IP experiments .

• Proximity ligation assays: Combine SEMA6D antibodies with antibodies against suspected binding partners to visualize protein-protein interactions in situ with subcellular resolution.

• FRET/BRET analysis: Label SEMA6D and binding partners with appropriate fluorophores or bioluminescent tags to monitor real-time interactions in live cells.

• Pull-down assays: Use recombinant SEMA6D domains (especially the semaphorin domain) to identify novel binding partners, followed by verification with co-IP using full-length protein.

• Cross-linking studies: Employ chemical cross-linkers to stabilize transient interactions before immunoprecipitation with SEMA6D antibodies.

These methodological approaches can help elucidate the molecular mechanisms underlying SEMA6D's diverse functions in neuronal guidance and cancer progression.

What emerging technologies could enhance SEMA6D research in the future?

Emerging technologies with potential to significantly advance SEMA6D research include:

• Single-cell analysis: Technologies like single-cell RNA-seq could reveal cell-type specific expression patterns of SEMA6D isoforms in heterogeneous tissues like tumors and neural tissues.

• CRISPR-based approaches: CRISPR/Cas9 genome editing can enable precise modification of SEMA6D domains to study structure-function relationships and isoform-specific roles.

• Advanced imaging techniques: Super-resolution microscopy combined with HRP-conjugated antibodies could provide nanoscale visualization of SEMA6D distribution and clustering during signaling events.

• Organoid and patient-derived xenograft models: These systems could better recapitulate the physiological context of SEMA6D function in development and disease compared to traditional cell lines.

• AI-driven protein structure prediction: Tools like AlphaFold could provide insights into the three-dimensional structure of SEMA6D domains and their interaction interfaces with binding partners.

What are the key considerations for developing novel functional assays for SEMA6D activity?

When developing functional assays for SEMA6D activity, researchers should consider:

• Physiological relevance: Design assays that reflect SEMA6D's known functions in axon guidance, migration, and cell adhesion.

• Isoform specificity: Ensure assays can distinguish between the seven splice variants which may have different functional outputs .

• Quantitative readouts: Develop high-throughput, quantifiable assays (e.g., automated neurite outgrowth analysis, cell migration tracking) for reproducible results.

• Context dependency: Account for SEMA6D's different roles across tissue types by including appropriate co-receptors and signaling partners in reconstituted systems .

• Validation controls: Include positive and negative controls with known SEMA6D activity modulation, such as domain-specific antibodies or competitive peptides.

How can comprehensive multi-omics approaches advance our understanding of SEMA6D biology?

Multi-omics approaches for advancing SEMA6D research should integrate:

• Genomics: Analysis of SEMA6D genetic variants, promoter regions, and alternative splicing regulation across populations and disease states.

• Transcriptomics: RNA-seq to identify co-regulated genes and pathways, potentially revealing new functional connections as demonstrated in ccRCC research .

• Proteomics: Mass spectrometry-based approaches to identify post-translational modifications and protein-protein interaction networks of SEMA6D.

• Metabolomics: Investigation of downstream metabolic changes resulting from SEMA6D signaling activity in different cellular contexts.

• Spatial transcriptomics/proteomics: Mapping SEMA6D expression patterns with spatial resolution in tissues to understand its role in complex multicellular environments.