SEMA3C Antibody, HRP conjugated

What is SEMA3C and why is it important in cancer research?

SEMA3C (Semaphorin 3C) is a member of the semaphorin family of secreted proteins that have been identified as novel tumor-associated factors. Previous studies have demonstrated that in the tumor microenvironment, SEMA3C promotes endotheliocyte migration, cancer metastasis, and angiogenesis. The deletion of SEMA3C has been shown to suppress tumorigenesis and angiogenesis, making it a protein of significant interest for cancer research . SEMA3C has specifically been implicated in breast cancer cell proliferation and migration, with studies showing that silencing SEMA3C expression in MCF-7 breast cancer cells significantly suppresses both proliferation and migration .

What applications are SEMA3C antibodies typically used for?

SEMA3C antibodies are employed in multiple research applications, including:

• Western blotting (WB) - For detecting and quantifying SEMA3C protein expression levels

• Enzyme-linked immunosorbent assay (ELISA) - For measuring SEMA3C concentrations

• Immunohistochemistry (IHC) - For visualizing SEMA3C in tissue sections

• Immunocytochemistry (ICC) - For localizing SEMA3C in cultured cells

• Immunofluorescence (IF) - For fluorescent visualization of SEMA3C

• Immunoprecipitation (IP) - For isolating SEMA3C protein complexes

The choice of application depends on the specific research question and experimental design. For protein expression analysis in cell lysates or tissue homogenates, Western blotting with SEMA3C antibodies is particularly effective.

How do I properly prepare samples for Western blotting with SEMA3C antibodies?

For optimal Western blot results with SEMA3C antibodies, follow this methodological approach:

• Harvest cells (e.g., MCF-7) and lyse in RIPA buffer

• Determine protein concentration using a BCA Protein Assay kit

• Load approximately 50 μg of protein per lane on 8% SDS-PAGE gels

• Transfer proteins to PVDF membranes

• Block membranes with 5% bovine serum albumin for 1 hour at room temperature

• Incubate with primary SEMA3C antibody (typical dilution: 1:800) overnight at 4°C

• Wash three times with TBS-Tween-20 (TBST)

• Incubate with HRP-conjugated secondary antibody for 2 hours at room temperature

• Wash again with TBST

• Detect protein bands using an enhanced chemiluminescence assay kit

• Analyze band intensity using software such as ImageJ, using GAPDH as an internal control

This protocol has been validated for detecting SEMA3C in breast cancer cell lines and can be adapted for other cell types.

What are the advantages of using HRP-conjugated SEMA3C antibodies versus unconjugated primary antibodies?

Using HRP-conjugated SEMA3C antibodies offers several methodological advantages:

• Streamlined workflow: Eliminates the need for a secondary antibody incubation step, reducing total assay time by approximately 2-3 hours

• Reduced cross-reactivity: Minimizes potential non-specific binding that can occur with secondary antibodies

• Improved signal-to-noise ratio: Direct conjugation can provide cleaner results with less background

• Quantification reliability: More direct relationship between signal intensity and protein quantity

• Multiplexing capability: Facilitates simultaneous detection of multiple proteins when combined with antibodies conjugated to different enzymes

What controls should be included when validating SEMA3C antibody specificity?

Proper validation of SEMA3C antibody specificity requires the following controls:

For SEMA3C knockdown validation, researchers have successfully used siRNA approaches with three different sequences targeting SEMA3C, achieving knockdown efficiencies of 47.37±6.02%, 50.87±4.61%, and 65.27±3.15% at the protein level, as measured by Western blot analysis .

How can I troubleshoot weak or absent signals when using SEMA3C antibodies in Western blots?

When encountering weak or absent signals with SEMA3C antibodies in Western blots, consider this systematic troubleshooting approach:

• Sample preparation issues:

  • Ensure proper cell lysis using RIPA buffer

  • Verify protein concentration using BCA assay

  • Increase loaded protein amount to 50-75 μg

  • Add protease inhibitors to prevent degradation

• Technical parameters:

  • Use 8% SDS-PAGE gels for optimal separation

  • Extend primary antibody incubation to overnight at 4°C

  • Optimize antibody dilution (try 1:500 to 1:1,000 range)

  • Increase exposure time during detection

• Antibody-specific factors:

  • Verify antibody reactivity matches your species (human, mouse, rat)

  • Confirm antibody recognizes the correct SEMA3C domain

  • Try alternative SEMA3C antibodies targeting different epitopes

  • For HRP-conjugated antibodies, check enzyme activity

• Detection system:

  • Use high-sensitivity ECL substrate for low abundance targets

  • Ensure proper membrane transfer using Ponceau S staining

  • Try fluorescent secondary antibodies as an alternative approach

How can SEMA3C antibodies be used to investigate SEMA3C-mediated cell signaling pathways?

SEMA3C antibodies can be employed in multiple sophisticated approaches to investigate signaling pathways:

• Subcellular fractionation studies:

  • Separate cytosolic and nuclear fractions of cells

  • Use SEMA3C antibodies to track protein localization

  • Correlate with β-catenin nuclear localization, as SEMA3C has been shown to significantly influence nuclear fraction of β-catenin in cellular fractionation assays

• Co-immunoprecipitation (Co-IP):

  • Use SEMA3C antibodies to pull down protein complexes

  • Perform Western blot analysis on precipitated samples

  • Identify binding partners in the Wnt signaling pathway

• Chromatin immunoprecipitation (ChIP):

  • Apply after treating cells with formaldehyde to crosslink proteins to DNA

  • Precipitate SEMA3C-associated transcription factors

  • Analyze binding to Wnt target gene promoters

• Rac1 activation assays:

  • SEMA3C has been shown to activate Rac1 to facilitate β-catenin translocation

  • Use SEMA3C antibodies to investigate this relationship in pull-down assays

  • Correlate with downstream Wnt target gene expression

Research has demonstrated that SEMA3C signaling can drive Wnt signaling through Rac1 activation, leading to β-catenin nuclear accumulation independent of Wnt ligand binding, representing an alternative activation mechanism for the canonical Wnt pathway .

What are the most effective approaches for studying SEMA3C cleavage by proteases like ADAMTS1?

Investigating SEMA3C cleavage by ADAMTS1 requires sophisticated experimental approaches:

• In vitro cleavage assays:

  • Incubate recombinant SEMA3C with purified ADAMTS1

  • Analyze cleavage products by Western blotting using domain-specific SEMA3C antibodies

  • Map cleavage sites through mass spectrometry analysis

• Cell-based cleavage monitoring:

  • Co-culture cells expressing SEMA3C with cells overexpressing ADAMTS1

  • Collect conditioned media and cell lysates at various time points

  • Analyze SEMA3C fragments using antibodies targeting different domains

  • Compare migration patterns of fragments with predicted molecular weights

• Functional consequences analysis:

  • Using HUVEC migration assays as a readout

  • Compare effects of intact versus cleaved SEMA3C

  • Research has shown that, unlike semaphorins 3A and 3B which induce repulsive signals, semaphorin 3C does not repel HUVECs, suggesting different functional effects

• Mutagenesis studies:

  • Generate SEMA3C mutants with altered predicted cleavage sites

  • Determine resistance to ADAMTS1-mediated cleavage

  • Assess functional consequences on cell migration and proliferation

Studies have revealed that while cells expressing semaphorins 3A or 3B repel HUVECs, cells expressing semaphorin 3C did not show this effect, suggesting either no impact on migration or potentially an attractive rather than repulsive signal .

How can SEMA3C antibodies be used to investigate the relationship between SEMA3C and the Wnt signaling pathway?

SEMA3C antibodies can be instrumental in elucidating the complex relationship between SEMA3C and Wnt signaling through these methodological approaches:

• Dual immunofluorescence staining:

  • Use SEMA3C antibodies in conjunction with β-catenin antibodies

  • Quantify nuclear β-catenin localization in relation to SEMA3C expression

  • Research has shown that knockdown of SEMA3C in GSCs (glioma stem cells) reduced the fraction of cells exhibiting nuclear β-catenin by at least 50%

• TCF/LEF reporter assays:

  • Transfect cells with TCF/LEF luciferase reporter constructs

  • Manipulate SEMA3C expression through knockdown or overexpression

  • Measure changes in Wnt pathway activation via luciferase activity

  • Combine with Wnt inhibitors (e.g., LGK974) to assess pathway independence

• Gene expression analysis:

  • Perform RT-qPCR for Wnt target genes after SEMA3C manipulation

  • Use SEMA3C antibodies to confirm knockdown efficiency

  • Correlate expression levels with Wnt target activation

• Therapeutic targeting studies:

  • Test combined inhibition of SEMA3C and Wnt pathways

  • Measure effects on cancer cell self-renewal capacity

  • Research has demonstrated that silencing both SEMA3C and TCF1 led to the greatest reduction in GSC self-renewal capacity compared to single knockdown, suggesting synergy with dual inhibition

Research indicates that SEMA3C-dependent Wnt signaling can occur despite suppression of Wnt ligand secretion, suggesting SEMA3C drives Wnt signaling independent of Wnt ligand binding, which has significant implications for resistance to Wnt inhibitors in cancer treatment .

What are the current limitations of SEMA3C antibodies and how might future developments address them?

Current limitations of SEMA3C antibodies and potential future improvements include:

• Specificity concerns:

  • Current challenge: Some antibodies may cross-react with other semaphorin family members

  • Future direction: Development of epitope-mapped antibodies targeting unique SEMA3C regions

  • Potential solution: Production of monoclonal antibodies against unique peptide sequences

• Functional blocking capacity:

  • Current challenge: Most antibodies are detection-oriented rather than function-blocking

  • Future direction: Design of antibodies specifically targeting receptor-binding domains

  • Application: These could serve as therapeutic tools beyond research reagents

• Post-translational modification detection:

  • Current challenge: Limited ability to distinguish different SEMA3C forms (cleaved vs. intact)

  • Future direction: Development of modification-specific antibodies (phosphorylation, glycosylation)

  • Benefit: Would enable more nuanced understanding of SEMA3C regulation

• Technological limitations:

  • Current challenge: Sensitivity limitations in detecting low-abundance SEMA3C

  • Future direction: Enhanced detection technologies like proximity ligation assays

  • Impact: Would allow visualization of protein interactions in situ

The development of these next-generation antibodies would significantly advance our understanding of SEMA3C biology and its role in cancer progression .

How can researchers optimize dual staining protocols using SEMA3C antibodies with other cancer biomarkers?

Optimizing dual staining protocols with SEMA3C antibodies requires careful consideration of several technical factors:

• Antibody compatibility planning:

  • Select SEMA3C antibodies from different host species than other target antibodies

  • If using same-species antibodies, consider directly conjugated primary antibodies

  • Verify non-overlapping epitopes when using multiple antibodies against SEMA3C

  • Example: Pair rabbit anti-SEMA3C with mouse anti-β-catenin antibodies

• Sequential staining protocol:

  • Apply heat-mediated antigen retrieval (optimal for SEMA3C detection)

  • Block with 5% BSA or serum matching secondary antibody host

  • Incubate with first primary antibody (SEMA3C) overnight at 4°C

  • Apply first secondary antibody (2 hours at room temperature)

  • Block again to prevent cross-reactivity

  • Apply second primary and secondary antibodies

  • Use DAPI (1:1000) for nuclear counterstaining

• Signal separation strategies:

  • Use fluorophores with well-separated emission spectra

  • Consider spectral unmixing for closely overlapping signals

  • When using HRP-conjugated antibodies, employ sequential chromogenic detection with different substrates

• Validation approach:

  • Always include single-stained controls

  • Use SEMA3C-knockdown samples as negative controls

  • Compare staining patterns with published SEMA3C localization data

This methodological approach has been effective for visualizing the relationship between SEMA3C expression and β-catenin nuclear localization in cancer cells .

What novel therapeutic approaches might emerge from a deeper understanding of SEMA3C function in cancer?

Research with SEMA3C antibodies is revealing several promising therapeutic approaches:

• RNA interference-based therapies:

  • Small interfering RNA (siRNA) targeting SEMA3C has shown significant efficacy

  • In MCF-7 breast cancer cells, SEMA3C siRNA achieved 65.27±3.15% knockdown efficiency

  • This resulted in significantly reduced cell proliferation (55.12±5.03% of control at 72h)

  • Migration was similarly inhibited (104.71±3.01 migrated cells vs. 198.16±9.07 in control)

• Dual pathway inhibition strategies:

  • Combined targeting of SEMA3C and Wnt pathways shows synergistic effects

  • Silencing both SEMA3C and TCF1 produced greater reduction in cancer stem cell self-renewal

  • This approach could overcome resistance to single-pathway inhibition

• Antibody-based therapeutic approaches:

  • Function-blocking antibodies targeting SEMA3C interaction with receptors

  • Antibody-drug conjugates for targeted delivery to SEMA3C-expressing tumors

  • Bi-specific antibodies targeting SEMA3C and immune checkpoint proteins

• Rational drug development:

  • Small molecule inhibitors disrupting SEMA3C-Rac1-β-catenin axis

  • Peptide mimetics interfering with SEMA3C signaling

  • Proteolysis-targeting chimeras (PROTACs) for SEMA3C degradation

These approaches are particularly promising as SEMA3C signaling can drive Wnt activation independent of Wnt ligands, potentially overcoming limitations of current Wnt inhibitors in clinical development .

How should researchers design rigorous experiments to validate SEMA3C antibody specificity?

A comprehensive validation strategy for SEMA3C antibodies should include:

• Genetic knockdown/knockout validation:

  • Implement siRNA knockdown using multiple sequences targeting SEMA3C

  • Validate knockdown efficiency at both mRNA level (RT-qPCR) and protein level (Western blot)

  • Compare multiple siRNA sequences (as done in previous research showing efficiencies of 47.37±6.02%, 50.87±4.61%, and 65.27±3.15%)

  • Include CRISPR/Cas9 knockout cells as gold-standard negative controls

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins of related semaphorins (SEMA3A, SEMA3B, etc.)

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

  • Compare reactivity across multiple species if cross-species reactivity is claimed

• Epitope mapping:

  • Use peptide arrays covering the entire SEMA3C sequence

  • Identify precise epitope recognition sites

  • Verify epitope conservation across target species

• Application-specific validation:

  • For each application (WB, ELISA, IHC, etc.), perform specific validations

  • Include appropriate positive controls (MCF-7 cells express detectable SEMA3C)

  • Document optimal conditions (antibody dilution, incubation time, temperature)

The most rigorous approach combines these methods, with genetic manipulation serving as the cornerstone of validation.

What considerations are important when selecting between different SEMA3C antibodies for specific research applications?

When selecting SEMA3C antibodies for specific applications, researchers should consider:

The search results indicate 82 SEMA3C Western Blot antibodies are available across 19 suppliers, with various options for reactivity (human, mouse, rat), formats (conjugated, unconjugated), and applications . This diversity allows researchers to select antibodies optimized for their specific experimental needs.

How can researchers accurately interpret SEMA3C expression data in the context of cancer heterogeneity?

Interpreting SEMA3C expression in heterogeneous cancer samples requires sophisticated analytical approaches:

• Multi-scale analysis strategy:

  • Tissue level: Use SEMA3C IHC on whole tumor sections to map expression patterns

  • Cellular level: Employ dual IHC/IF to correlate SEMA3C with cell-type markers

  • Subcellular level: Analyze nuclear vs. cytoplasmic localization

  • This comprehensive approach reveals expression patterns missed by bulk analysis

• Tumor microenvironment considerations:

  • Determine whether SEMA3C is expressed by tumor cells, stromal cells, or both

  • Correlate with markers of cancer-associated fibroblasts, immune cells, and endothelial cells

  • Assess relationship to vascular structures (SEMA3C influences endothelial cell behavior)

• Quantitative image analysis methods:

  • Apply digital pathology approaches to quantify SEMA3C expression

  • Use machine learning algorithms to identify expression patterns across tumor regions

  • Correlate expression with histopathological features

  • Compare with β-catenin localization patterns to assess Wnt pathway activation

• Integration with molecular profiling:

  • Correlate IHC/IF findings with RNA-seq data from matched samples

  • Assess relationship between protein and mRNA levels

  • Evaluate SEMA3C expression in relation to molecular subtypes

  • Studies have shown strong concordance between SEMA3C and TCF1 expression in human glioblastoma

This multi-modal approach provides context for interpreting SEMA3C expression beyond simple positive/negative classification.