SEMA3C Antibody

What is SEMA3C and what protein characteristics are important when selecting an antibody?

SEMA3C (Semaphorin 3C) is a secreted protein belonging to the semaphorin family. When selecting antibodies, consider these key characteristics:

• Molecular Weight: Approximately 85.2 kDa in its full-length form, though observed at ~70 kDa in some Western blot applications

• Alternative Nomenclature: Also known as SEMAE, SemE, and sema E

• Domain Structure: Contains sema domain, immunoglobulin domain (Ig), and short basic domain

• Species Homology: Human SEMA3C has orthologs in canine, porcine, monkey, mouse, and rat models, making cross-species reactivity an important consideration

What are the typical applications for SEMA3C antibodies in research?

SEMA3C antibodies can be used in multiple experimental techniques:

What species reactivity should researchers consider when selecting SEMA3C antibodies?

Most commercial SEMA3C antibodies show reactivity with:

• Human samples (most common)

• Mouse samples (frequently used in developmental studies)

• Rat samples (less common but available)

When planning cross-species experiments, verify that the epitope region is conserved across species. Some antibodies show high cross-reactivity due to the significant homology between human and rodent SEMA3C sequences .

How should researchers optimize SEMA3C detection in immunohistochemistry applications?

For optimal SEMA3C detection in tissue sections:

• Fixation Protocol:

  • For frozen sections: 4% paraformaldehyde overnight provides good antigen preservation while maintaining epitope accessibility

  • For paraffin sections: Standard formalin fixation works well, but antigen retrieval is critical

• Staining Optimization:

  • Concentration: Start with 5-20 µg/mL for most antibodies

  • Background reduction: Extended blocking (1 hour) with 5% normal serum from the secondary antibody host species

  • Signal amplification: Consider using HRP-DAB detection systems for chromogenic visualization in tissues with low expression

• Controls:

  • Positive control: Developing mouse embryo tissue (13-15 d.p.c.), particularly developing muscle and neural crest cells

  • Negative control: SEMA3C knockout tissue if available, or omission of primary antibody

R&D Systems reported successful staining in mouse embryo using their antibody (catalog # MAB1728) at 1.7 µg/mL with overnight incubation at 4°C, which showed specific localization to developing muscle cells .

What are the critical considerations for Western blot detection of SEMA3C?

For successful Western blot detection of SEMA3C:

• Sample Preparation:

  • Include protease inhibitors to prevent degradation

  • Mouse heart tissue provides a reliable positive control

• Technical Parameters:

  • Protein loading: 20-50 µg of total protein per lane

  • Antibody dilution: 1:500-1:2000 for most antibodies

  • Expected band: Primary band at ~70-85 kDa, though processing/cleavage products may appear

• Troubleshooting:

  • Multiple bands may indicate post-translational modifications or proteolytic processing

  • Absence of signal in positive control samples may require extended exposure times

  • Non-specific binding can be reduced with more stringent washing conditions

According to validation data, observed molecular weight is often around 70 kDa despite the calculated molecular weight of 85 kDa, likely due to proteolytic processing or migration behavior .

How does SEMA3C expression change in disease models, and what methodological approaches are best for studying these changes?

SEMA3C shows significant expression changes in disease contexts:

• Inflammatory Conditions:

  • Upregulated after spinal cord injury (SCI)

  • Predominantly expressed by M1-type (proinflammatory) microglia/macrophages

  • Detected using immunofluorescence co-localization with Iba-1 (microglia marker)

• Cancer Models:

  • Associated with tumor progression in pancreatic, gastric, prostate, breast cancer, and glioma

  • Paradoxically reported as both oncogenic and tumor-suppressive depending on context

  • Methods: Combine IHC with patient outcome data for clinical correlation

• Methodology for Disease Studies:

  • Multi-method approach combining Western blot, qPCR, and immunostaining

  • Time-course experiments to track expression changes (crucial for injury models)

  • Cell-type specific markers for co-localization studies

Recent research showed that SEMA3C protein expression increased over time after spinal cord injury, consistent with qPCR analysis, suggesting a role in neuroinflammatory processes .

What experimental setup is needed to study SEMA3C interaction with its receptors and signaling pathways?

To investigate SEMA3C signaling mechanisms:

• Receptor Interaction Studies:

  • Co-immunoprecipitation assays with SEMA3C antibodies to pull down receptor complexes

  • Proximity ligation assays to visualize interaction with plexin family receptors in situ

  • Molecular docking software to predict ligand-receptor interactions (as performed for SEMA3C-RAGE with a binding score of -562)

• Signaling Pathway Analysis:

  • RAGE/NF-κB pathway activation: Measure phospho-NF-κB p65 and NLRP3 levels

  • Downstream effects: Quantify proinflammatory cytokines (TNF-α, IL-6, IL-1β) by ELISA

  • Inhibition studies: Use pathway blockers (e.g., FPS-ZM1 for RAGE pathway) to confirm signaling specificity

• Functional Validation:

  • Cell polarization assays: Assess M1/M2 macrophage/microglia markers following SEMA3C treatment

  • Pathway verification: Double immunostaining with Arg-1(M2)/CD68 to characterize polarization states

Recent findings indicate that SEMA3C interacts with RAGE through multiple hydrogen bonds, such as between His537 of SEMA3C and Arg177 of RAGE, activating inflammatory signaling in microglia/macrophages .

What are common sources of non-specific staining when using SEMA3C antibodies, and how can they be addressed?

Addressing non-specific staining challenges:

• Common Sources of Background:

  • One researcher reported non-specific staining on arterial lining of heart tissue using a SEMA3C antibody at 1:200 dilution

  • Cross-reactivity with other semaphorin family members due to conserved domains

  • Endogenous peroxidase activity in tissue sections

  • Fc receptor binding in immune cells

• Optimization Strategies:

  • Validation in knockout models to confirm specificity (as demonstrated in Sema3C-KO mice)

  • Titration experiments to determine optimal antibody concentration

  • Extended blocking steps (5% serum, 1-2 hours)

  • Pre-adsorption with recombinant protein for polyclonal antibodies

  • Use alternative detection methods (fluorescence vs. chromogenic)

• Control Experiments:

  • Include isotype controls at equivalent concentrations

  • Compare patterns with multiple antibodies targeting different SEMA3C epitopes

  • Use competing peptides to demonstrate specificity

How should researchers approach antibody validation to ensure specificity for SEMA3C?

A comprehensive validation strategy includes:

• Genetic Approaches:

  • Testing in knockout/knockdown models (as demonstrated in Sema3C-KO mice where immunofluorescence confirmed no detection of nonspecific antigens)

  • siRNA or CRISPR-mediated downregulation followed by antibody testing

• Biochemical Validation:

  • Western blot should show expected molecular weight (70-85 kDa)

  • Immunoprecipitation followed by mass spectrometry identification

  • Peptide competition assays to confirm epitope specificity

• Cross-Platform Verification:

  • Correlation between protein detection (immunostaining) and mRNA expression (in situ hybridization)

  • Comparison of multiple antibodies targeting different epitopes

  • Recombinant protein controls expressing tagged SEMA3C

One study validated SEMA3C expression using complementary approaches: "Expression level of SEMA3C protein at different time points was detected by Western blot. The Western blot result showed a marked increase in SEMA3C expression over time, which was consistent with real-time qPCR analysis" .

What are the key considerations when designing experiments to study the dual roles of SEMA3C in normal development versus pathological conditions?

When investigating SEMA3C's contextual roles:

• Developmental Studies:

  • Timing is critical: SEMA3C shows dynamic expression during embryogenesis

  • Tissue-specific expression: Focus on cardiovascular and nervous systems

  • Techniques: Whole-mount immunostaining and in situ hybridization on developmental series

  • Controls: Age-matched wild-type vs. knockout embryos

• Pathological Contexts:

  • Cancer: SEMA3C has been reported as both tumor-promoting and tumor-suppressive

  • Inflammation: Primarily proinflammatory in neurological injury

  • Experimental design must account for these contradictory roles

• Methodological Approach:

  • Combine functional assays with expression studies

  • Distinguish full-length from processed forms (which may have different functions)

  • Use domain-specific antibodies to identify cleaved fragments

  • Time-course experiments to track dynamic changes

Research indicates that "full-length SEMA3C has also been reported as a tumor suppressor factor by suppressing tumor lymphangiogenesis and metastasis" while also being "associated with tumor progression and poor prognosis across multiple tumor types" . This apparent contradiction requires careful experimental design with appropriate controls and multiple methodological approaches.

How can researchers effectively use SEMA3C antibodies to study its role in neuroinflammation?

To investigate SEMA3C in neuroinflammatory contexts:

• Experimental Models:

  • Spinal cord injury (SCI) provides a well-characterized model

  • BV2 microglial cell culture for in vitro mechanistic studies

  • LPS/IFN-γ stimulation to induce M1 polarization and SEMA3C upregulation

• Analytical Approach:

  • Time-course studies: Monitor SEMA3C expression at different post-injury timepoints

  • Cell-type analysis: Co-immunostaining with Iba-1 (microglia) and additional markers

  • Pathway investigation: Assess RAGE/NF-κB activation and downstream mediators

• Functional Studies:

  • Manipulate SEMA3C levels through recombinant protein addition or knockdown

  • Measure inflammatory cytokines (TNF-α, IL-6, IL-1β) by ELISA

  • Assess microglial polarization using M1/M2 markers

  • Implement pathway inhibitors to confirm mechanistic relationships

Recent research demonstrated that "SEMA3C treatment upregulated the levels of RAGE, NF-κB p65, phospho-NF-κB p65, and NLRP3, all key factors involved in the RAGE-signaling axis," providing a mechanism for SEMA3C's proinflammatory effects in neurological injury .

What methodologies are effective for studying the relationship between SEMA3C and cancer progression?

For cancer-related SEMA3C research:

• Patient-Derived Samples:

  • Tissue microarrays for IHC assessment across tumor stages

  • Correlation of SEMA3C expression with clinical outcomes

  • Comparison between tumor and adjacent normal tissues

• Cell Line Models:

  • MCF-7 breast cancer cells show notable SEMA3C expression (localized to cytoplasm)

  • Western blot analysis with careful quantification

  • Immunocytochemistry using optimized protocols (10 µg/mL antibody concentration, 3-hour incubation)

• Functional Approaches:

  • Migration and invasion assays following SEMA3C modulation

  • Study of both autocrine and paracrine effects

  • Analysis of full-length versus processed forms, which may have distinct functions

• Recommended Controls:

  • Multiple cell lines with varying SEMA3C expression levels

  • Genetic knockdown/knockout verification

  • Recombinant SEMA3C treatment to confirm phenotypes

The dual role of SEMA3C in cancer progression requires careful experimental design and multiple methodological approaches to fully characterize its context-dependent functions .

What are the best practices for studying SEMA3C interactions with plexin receptors and downstream signaling?

To effectively investigate SEMA3C-receptor interactions:

• Binding Studies:

  • Co-immunoprecipitation with plexin family members

  • Surface plasmon resonance for binding kinetics

  • Proximity ligation assays for in situ interaction visualization

  • Computational approaches: "Molecular docking software to predict the ligand-receptor interaction" as demonstrated for SEMA3C-RAGE

• Signaling Analysis:

  • Phosphorylation studies of key pathway components

  • Real-time signaling using fluorescent reporters

  • Inhibitor studies to confirm pathway specificity

  • Genetic approaches (dominant-negative receptors, pathway component knockdowns)

• Functional Readouts:

  • Growth cone collapse assays for neuronal effects

  • Cell migration tracking following receptor manipulation

  • Gene expression changes using RNA-seq or targeted qPCR

  • Cytokine profiling in inflammatory models

• Technical Considerations:

  • Careful antibody selection to avoid interference with receptor binding sites

  • Use of tagged constructs (His, Fc) for tracking and purification

  • Domain-specific antibodies to distinguish different processing forms