SCN4B Antibody, FITC conjugated

What is SCN4B and why is it important for research?

SCN4B encodes the β4 protein, an auxiliary subunit of voltage-gated sodium channels (Nav) initially characterized in excitable tissues. Recent research has revealed its expression in normal epithelial cells and its function as a metastasis-suppressor gene in cancer biology. The β4 protein contains an extracellular immunoglobulin (Ig) domain, a transmembrane domain, and an intracellular C-terminus, each contributing to its biological functions. In cancer cells, reduced β4 expression increases RhoA activity, potentiating cell migration, invasiveness, and metastatic spreading through the acquisition of an amoeboid–mesenchymal hybrid phenotype . Additionally, SCN4B plays a critical role in immune system development, particularly in the positive selection of CD4+ T cells through its extracellular Ig domain .

What applications are suitable for FITC-conjugated SCN4B antibodies?

FITC-conjugated SCN4B antibodies are particularly valuable for fluorescence-based applications including:

• Flow cytometry for quantitative analysis of SCN4B expression in cell populations

• Immunofluorescence microscopy for subcellular localization studies

• FACS (Fluorescence-Activated Cell Sorting) for isolating SCN4B-expressing cells

• Live-cell imaging to monitor dynamic changes in SCN4B distribution

The antibody shows reliable reactivity with human, mouse, and rat samples, making it suitable for comparative studies across these species . For optimal results in flow cytometry, a concentration of 0.25 μg per 10^6 cells in a 100 μl suspension is recommended, although titration may be necessary for each experimental system .

How should FITC-conjugated SCN4B antibodies be stored to maintain fluorescence activity?

To preserve both antibody integrity and FITC fluorescence:

• Store at -20°C in the dark to prevent photobleaching

• Aliquot to avoid repeated freeze-thaw cycles

• Maintain in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Keep aliquots stable for up to one year after shipment when properly stored

• For short-term storage (1-2 weeks), 4°C is acceptable if protected from light

Smaller aliquots (20μl) containing 0.1% BSA help stabilize the antibody during storage and prevent non-specific binding during applications .

What controls should be included when using FITC-conjugated SCN4B antibodies?

For rigorous experimental design, include these essential controls:

These controls help distinguish true SCN4B signals from potential artifacts, particularly important when studying tissues with complex autofluorescence profiles or when performing quantitative analyses .

How can FITC-conjugated SCN4B antibodies be optimized for dual immunofluorescence studies?

For multiplexed detection with other targets, consider these optimization strategies:

• Select compatible fluorophores to minimize spectral overlap (FITC emission peaks at ~520 nm)

• When pairing with red-emitting fluorophores (e.g., Cy3, Cy5), sequence the staining protocol to:

  • Apply the FITC-conjugated SCN4B antibody first

  • Fix with 2% paraformaldehyde to stabilize the binding

  • Proceed with the second primary-secondary antibody pair

For samples with high autofluorescence in the FITC channel, consider:

• Pre-treatment with 0.1% Sudan Black B in 70% ethanol for 20 minutes

• Photobleaching treatments prior to antibody application

• Using spectral unmixing during image acquisition and analysis

These approaches have successfully resolved SCN4B co-localization with voltage-gated sodium channel α subunits and RhoA in cancer cell lines, revealing functional interactions relevant to metastatic behavior .

What methodological considerations are important when using FITC-conjugated SCN4B antibodies for detecting metastasis-suppressor activity?

When investigating SCN4B's role as a metastasis suppressor, consider this experimental workflow:

• Sample preparation optimization:

  • For breast cancer tissues, use formalin-fixed, paraffin-embedded sections with antigen retrieval using TE buffer pH 9.0

  • For fresh tumor samples, prepare single-cell suspensions using non-enzymatic dissociation to preserve membrane epitopes

• Expression correlation analysis:

  • Quantify SCN4B levels across tumor grades (I-III) using standardized mean fluorescence intensity

  • Compare against clinical outcomes and established metastasis markers

• Functional validation experiments:

  • Complement antibody staining with RhoA activity assays (FRET-based or pull-down)

  • Correlate SCN4B expression with cell migration parameters in wound healing or transwell assays

Research demonstrates that reduced β4 protein levels in breast cancer biopsies correlate with high-grade primary and metastatic tumors, making SCN4B a potential prognostic marker .

How can FITC-conjugated SCN4B antibodies be used to investigate Na+ channel-independent functions of the β4 protein?

To specifically examine Nav-independent functions:

• Domain-specific approach:

  • Use domain-blocking experiments with SCN4B-Ig fusion proteins to compete with specific regions

  • Compare results with TTX (tetrodotoxin) treatment to differentiate between channel-dependent and independent effects

  • Include experimental groups with expression of truncated SCN4B (ΔN-ter or ΔC-ter) to isolate domain-specific functions

• Combined techniques for mechanism determination:

  • Flow cytometry with FITC-SCN4B antibody for expression quantification

  • Proximity ligation assays to detect β4-RhoA interactions

  • Pull-down assays to measure RhoA, Rac1, and Cdc-42 activities

Research has established that SCN4B/β4 inhibits RhoA activation independently of Nav function, with the intracellular C-terminus playing a crucial role in this inhibition .

What are the technical considerations for using FITC-conjugated SCN4B antibodies in immune cell research?

For investigating SCN4B in T cell development and function:

• Thymocyte preparation protocols:

  • Harvest thymocytes in calcium-free media to prevent activation

  • Use gentle mechanical dissociation without enzymatic treatment

  • Maintain samples at 4°C throughout processing

• Calcium signaling experiments:

  • Combine FITC-SCN4B staining with ratiometric calcium indicators (Fura-2)

  • Implement real-time imaging with stimulation using:

    • gp250 (known positive selector for AND T cell receptors)

    • Interferon-γ to upregulate MHC class presentation

• Reaggregate culture methodologies:

  • Establish AND transgenic DP thymocyte cultures with thymic epithelial cells

  • Analyze SCN4B distribution before and after positive selection

  • Correlate with calcium flux measurements and CD4/CD8 lineage outcomes

The SCN4B extracellular domain is essential for positive selection by regulating the SCN5a pore in cis during thymocyte selection, offering insights into T cell development mechanisms .

How can discrepancies between observed and predicted molecular weights of SCN4B be resolved in western blotting?

The calculated molecular weight of SCN4B is 25 kDa, but it commonly appears at 37 kDa in western blots, causing potential confusion . This discrepancy may result from:

• Post-translational modifications:

  • N-glycosylation at predicted sites in the extracellular domain

  • Potential phosphorylation at serine/threonine residues

• Methodological approaches to resolve this discrepancy:

  • Include deglycosylation treatments (PNGase F) before electrophoresis

  • Run both reduced and non-reduced samples to assess disulfide bonding effects

  • Use gradient gels (4-20%) for better resolution of mid-range proteins

  • Include both positive controls and recombinant SCN4B protein standards

• Validation strategy:

  • Compare banding patterns between multiple anti-SCN4B antibodies

  • Confirm specificity through genetic knockdown (siRNA or shRNA)

  • Perform mass spectrometry analysis of the immunoprecipitated protein

These approaches help distinguish true SCN4B signal from potential cross-reactivity with other sodium channel β subunits, which share structural similarities .

What are the most effective protocols for detecting SCN4B in tissues with high background autofluorescence?

When working with tissues prone to autofluorescence (brain, heart, adipose):

• Pre-treatment options:

  • CuSO₄ (10mM in 50mM ammonium acetate buffer, pH 5.0) for 1 hour

  • 0.3% Sudan Black B in 70% ethanol for 20 minutes

  • 0.1% Sodium borohydride in PBS for 5 minutes (fresh solution)

• Acquisition strategies:

  • Implement spectral unmixing on confocal microscopes

  • Use time-gated detection to separate antibody fluorescence from autofluorescence

  • Consider super-resolution techniques (STED, STORM) for improved signal discrimination

• Analysis approaches:

  • Apply local background subtraction with appropriate controls

  • Implement machine learning algorithms to distinguish signal patterns

  • Use ratiometric analysis against known expression patterns

These methods have successfully detected SCN4B in challenging tissues including dorsal root ganglia, brain tissue, and cardiac samples where autofluorescence can otherwise mask specific signals .

How should researchers address potential epitope masking when investigating SCN4B interactions with other proteins?

When studying SCN4B protein interactions:

• Fixation considerations:

  • Mild fixation (2% PFA for 10 minutes) preserves epitope accessibility

  • Alcohol-based fixatives may better preserve membrane protein epitopes

  • Avoid glutaraldehyde, which can significantly mask epitopes

• Antigen retrieval options:

  • Test both citrate buffer (pH 6.0) and TE buffer (pH 9.0)

  • Optimize retrieval duration (10-30 minutes) based on tissue type

  • Consider non-heat retrieval methods for sensitive epitopes

• Blocking strategy:

  • Use 5% normal serum from the same species as secondary antibody

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization

  • Consider dual blocking with both serum and 1% BSA

• Detection approach:

  • Implement proximity ligation assays for protein interactions

  • Use epitope tagging in overexpression studies as alternative detection method

  • Apply FRET-based approaches for direct interaction studies

These techniques help detect important interactions between SCN4B and RhoA, which are crucial for understanding its metastasis-suppressor function .

How can FITC-conjugated SCN4B antibodies be utilized to study the amoeboid-mesenchymal transition in cancer cells?

The shift between mesenchymal and amoeboid migration modes is critical in metastasis. To investigate SCN4B's role:

• Experimental design for migration phenotyping:

  • Combine SCN4B-FITC labeling with F-actin visualization (phalloidin-TRITC)

  • Implement live-cell imaging with ECM degradation reporters

  • Quantify morphological parameters (circularity index, protrusion dynamics)

• Analysis of migration mode markers:

  • Correlate SCN4B expression with mesenchymal markers (MT1-MMP, cortactin)

  • Measure RhoA/ROCK pathway activation in SCN4B-positive versus negative populations

  • Assess focal adhesion dynamics using paxillin or vinculin co-staining

• 3D invasion assays:

  • Track SCN4B-FITC labeled cells in collagen matrices

  • Analyze invasion patterns (collective versus single-cell)

  • Compare migration speeds and paths between SCN4B-high and SCN4B-low populations

Research demonstrates that reducing β4 expression increases RhoA activity and promotes an amoeboid–mesenchymal hybrid phenotype, enhancing invasiveness through matrix remodeling capabilities combined with increased contractility .

What methodological approaches can determine if FITC-conjugated SCN4B antibodies affect the function of the target protein during live-cell experiments?

When using FITC-conjugated antibodies in live-cell applications:

• Functional interference assessment:

  • Compare migration rates between unlabeled and FITC-SCN4B labeled cells

  • Measure RhoA activity before and after antibody application

  • Assess calcium fluxes in labeled versus unlabeled populations

• Alternative labeling strategies:

  • Use Fab fragments instead of complete IgG to minimize crosslinking

  • Consider genetic tagging approaches (EGFP-SCN4B fusion) for validation

  • Implement pulse-chase experiments with different antibody concentrations

• Control experiments:

  • Include non-binding FITC-conjugated isotype controls

  • Test multiple epitope-targeting antibodies to identify minimal-interference options

  • Validate with knockdown/rescue experiments using siRNA and cDNA expression

These approaches help ensure that experimental observations reflect true biological phenomena rather than artifacts of antibody binding, particularly important when studying SCN4B's role in dynamic processes like cell migration .

How can FITC-conjugated SCN4B antibodies contribute to developing targeted therapies for metastatic cancer?

Building on SCN4B's role as a metastasis suppressor:

• Therapeutic target identification:

  • Use FITC-SCN4B antibodies to screen patient samples for expression levels

  • Correlate expression with treatment response and survival outcomes

  • Identify druggable interactions through co-localization studies

• Drug development strategies:

  • Screen for compounds that upregulate SCN4B expression

  • Develop mimetic peptides based on the C-terminal domain structure

  • Design RhoA pathway inhibitors specific to SCN4B-negative cancers

• Personalized medicine applications:

  • Establish SCN4B expression thresholds for treatment decisions

  • Monitor therapy-induced changes in SCN4B distribution

  • Combine with other metastasis markers for improved prognostic accuracy

Research has shown that SCN4B overexpression reduces cancer cell invasiveness and tumor progression, suggesting therapeutic potential in restoring its expression or function .

What innovative approaches can combine FITC-conjugated SCN4B antibodies with emerging technologies for single-cell analysis?

For cutting-edge single-cell research:

• Integration with single-cell technologies:

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) with FITC-SCN4B antibodies

  • Combine with mass cytometry (CyTOF) using metal-conjugated anti-FITC secondary antibodies

  • Apply for spatial transcriptomics with in situ hybridization

• Multiparametric analysis methodologies:

  • Develop computational pipelines for integrating protein expression with transcriptomic data

  • Implement machine learning algorithms to identify cell populations with distinct SCN4B functional states

  • Create reference atlases of SCN4B expression across cancer progression stages

• Dynamic analysis approaches:

  • Use microfluidic systems for real-time monitoring of SCN4B-expressing cells

  • Apply optogenetic tools in combination with SCN4B labeling

  • Implement biosensor technologies to correlate SCN4B localization with RhoA activity

These advanced approaches can reveal heterogeneity in SCN4B expression and function at single-cell resolution, potentially identifying resistant cell populations or novel therapeutic targets .