SCN4B Antibody, Biotin conjugated

What is the structure and functional domains of the SCN4B protein?

The SCN4B protein contains several key domains that contribute to its function:

• An extracellular immunoglobulin (Ig) domain that is critical for its function in regulating the SCN5a pore in cis

• A transmembrane domain

• An intracellular C-terminus domain

The amino acid region 31-162 appears to be particularly important for antibody recognition and contains significant functional elements . The extracellular Ig domain has been shown to be essential for positive selection by its regulation of the SCN5a pore . Research has also demonstrated that the C-terminus of SCN4B plays a critical role in preventing hyperactivated migration in cancer cells .

How do biotin-conjugated SCN4B antibodies differ from unconjugated versions?

Biotin-conjugated SCN4B antibodies contain a covalently attached biotin molecule that enables detection through avidin/streptavidin systems, offering several methodological advantages over unconjugated antibodies:

• Enhanced sensitivity in detection systems due to the strong biotin-avidin interaction

• Compatibility with multiple detection platforms including ELISA and streptavidin-based visualization systems

• Reduced background when compared to directly labeled antibodies

• Greater flexibility in experimental design, particularly for multi-labeling experiments

How can SCN4B antibodies be utilized to investigate the protein's role in cancer metastasis?

SCN4B has been identified as a metastasis-suppressor gene, making it a valuable target for cancer research. To investigate its role in metastasis:

• Tissue microarray analysis: Use immunohistochemistry with validated SCN4B antibodies to compare expression levels across normal tissues, primary tumors, and metastatic samples. Studies have shown that reduced β4 protein levels correlate with high-grade primary and metastatic tumors in breast cancer .

• Migration assays: After manipulating SCN4B expression (through knockdown or overexpression), measure changes in cancer cell migration. Researchers have demonstrated that reducing β4 expression increases RhoA activity and potentiates cell migration and invasiveness .

• Protein-protein interaction studies: Use co-immunoprecipitation with biotin-conjugated SCN4B antibodies to identify binding partners. Proximity ligation assays have indicated a close association between SCN4B/β4 protein and RhoA in cancer cells .

• Tumor growth analysis: In animal models, track how SCN4B expression affects primary tumor growth and metastatic spreading. Overexpression of SCN4B reduces cancer cell invasiveness and tumor progression .

These approaches can help elucidate the mechanisms by which SCN4B functions as a metastasis suppressor and potentially identify new therapeutic targets.

What methodological considerations are important when using biotin-conjugated SCN4B antibodies for immunohistochemistry?

When using biotin-conjugated SCN4B antibodies for immunohistochemistry (IHC), several methodological considerations are critical:

• Endogenous biotin blocking: Tissues, particularly liver, kidney, and many tumors, contain endogenous biotin that can cause high background. Pretreat sections with avidin-biotin blocking reagents before antibody application.

• Antigen retrieval optimization: For SCN4B detection, heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions.

• Antibody validation controls:

  • Positive control tissues known to express SCN4B (normal epithelial tissues)

  • Negative controls (primary antibody omission)

  • Absorption controls using the immunizing peptide

• Signal amplification systems: For low-abundance SCN4B detection, consider using tyramide signal amplification (TSA) or other amplification methods compatible with biotin-streptavidin systems.

• Multi-labeling considerations: When performing double or triple labeling, carefully select primary antibodies from different host species and use appropriate detection systems to prevent cross-reactivity.

The antibody concentration should be carefully titrated, with starting dilutions of approximately 1:1000 for Western blotting applications, and adjusted accordingly for IHC based on signal intensity and background levels .

What is the optimal protocol for using biotin-conjugated SCN4B antibodies in ELISA applications?

Optimized ELISA Protocol for Biotin-Conjugated SCN4B Antibodies:

• Plate coating:

  • Coat 96-well plates with capture antibody against your target protein

  • Incubate overnight at 4°C

  • Wash 3-5 times with washing buffer (PBS + 0.05% Tween-20)

• Blocking:

  • Block with 2-5% BSA in PBS for 1-2 hours at room temperature

  • Wash 3-5 times

• Sample addition:

  • Add samples and standards in appropriate dilution buffer

  • Incubate for 2 hours at room temperature or overnight at 4°C

  • Wash 3-5 times

• Biotin-conjugated SCN4B antibody addition:

  • Dilute biotin-conjugated SCN4B antibody (ABIN7169939) in dilution buffer

  • Add to wells and incubate for 1-2 hours at room temperature

  • Wash 3-5 times

• Detection:

  • Add streptavidin-HRP (1:10,000 to 1:20,000 dilution)

  • Incubate for 30-60 minutes at room temperature

  • Wash 5 times

  • Add TMB substrate and monitor color development

  • Stop reaction with 2N H₂SO₄

  • Read absorbance at 450nm

For optimal results, titrate the biotin-conjugated SCN4B antibody to determine the concentration that provides the best signal-to-noise ratio. The antibody has been specifically validated for ELISA applications with human samples and shows high specificity for amino acids 31-162 of the SCN4B protein .

How can SCN4B-Ig fusion proteins be generated and utilized in functional studies?

Generation and Application of SCN4B-Ig Fusion Proteins:

• Construction of expression vectors:

  • Design primers to amplify the extracellular domain of SCN4B (particularly the Ig domain)

  • Clone the amplified sequence into an expression vector containing an Ig Fc region

  • Verify the construct by sequencing

• Protein expression and purification:

  • Transfect mammalian cells (HEK293 or CHO cells) with the expression vector

  • Collect conditioned media containing secreted fusion protein

  • Purify using Protein A/G affinity chromatography

  • Verify purity by SDS-PAGE and specificity by Western blotting

• Functional applications:

  • Ligand identification: Use purified SCN4B-Ig fusion protein for pull-down assays to identify binding partners

  • Functional blocking: Apply SCN4B-Ig fusion protein to saturate potential ligands and study resulting functional effects

  • T-cell selection studies: Research has shown that SCN4B-Ig fusion protein can inhibit the positive selection of CD4+ T cells in vitro

• Calcium response assays:

  • Prepare pre-selected DP thymocytes

  • Treat with SCN4B-Ig fusion protein

  • Measure Ca²⁺ responses to determine the regulatory effects on SCN5a pore function

This approach has successfully demonstrated that the SCN4B extracellular domain is essential for positive selection by regulating the SCN5a pore in cis .

What are common sources of false positives/negatives when using biotin-conjugated SCN4B antibodies, and how can they be addressed?

Common Issues and Solutions:

For biotin-conjugated SCN4B antibodies specifically, ensure that the antibody preparation maintains recognition of the target epitope (amino acids 31-162) after conjugation, and verify specificity against human samples where it has been validated.

How should researchers interpret varied SCN4B expression patterns across different tissue types?

Interpreting SCN4B expression patterns requires understanding of its diverse roles across tissues:

• Normal vs. pathological expression:

  • In normal epithelial cells, SCN4B is expressed at consistent levels

  • Reduced expression in breast cancer biopsies correlates with high-grade primary and metastatic tumors

  • Expression patterns should be interpreted in the context of disease progression

• Tissue-specific functions:

  • In excitable tissues: primarily functions as an auxiliary subunit of voltage-gated sodium channels

  • In epithelial tissues: functions as a metastasis suppressor independent of sodium channels

  • In immune system: essential for positive selection of CD4+ T cells

• Quantification considerations:

  • Use digital image analysis for quantitative assessment of staining intensity

  • Establish tissue-specific expression baselines

  • Consider relative expression rather than absolute values when comparing across tissue types

• Subcellular localization:

  • Membrane localization indicates potential involvement in channel modulation

  • Cytoplasmic or nuclear localization may suggest alternative functions

  • Changes in subcellular distribution may correlate with pathological states

Research has shown that SCN4B expression can be evaluated through immunohistochemistry in tissue microarrays containing various tissue types, allowing for comparative analysis across normal, hyperplastic and dysplastic samples, as well as carcinomas of different grades .

How can researchers investigate the sodium channel-independent functions of SCN4B in cancer progression?

To investigate sodium channel-independent functions of SCN4B in cancer progression:

• Domain-specific constructs:

  • Generate truncated variants of SCN4B protein:

    • Full-length wild-type protein

    • N-terminus-truncated protein (lacking the Ig-like extracellular domain)

    • C-terminus-truncated protein

  • Express these variants in SCN4B-knockdown cells to determine which domains rescue the phenotype

• RhoA activity assays:

  • Measure RhoA activity using pull-down assays in cells with manipulated SCN4B expression

  • Research has shown that reduced SCN4B expression increases RhoA activity while decreasing Rac1 and Cdc-42 activities

  • Use proximity ligation assays to detect close association between SCN4B/β4 protein and RhoA

• Migration and invasion studies:

  • Perform migration assays with cells expressing different SCN4B constructs

  • Conduct invasion assays through extracellular matrix

  • Test the effect of myosin II inhibition (using blebbistatin) on invasiveness

• Matrix degradation analysis:

  • Analyze focalized Matrigel degradation as a measure of mesenchymal activity

  • Research has shown that overexpression of SCN4B/β4 protein reduces ECM proteolytic activity

These approaches have demonstrated that SCN4B functions independently from sodium channels in cancer cells, with its C-terminus playing a crucial role in preventing hyperactivated migration .

What methodological approaches can be used to study the interaction between SCN4B and RhoA signaling pathways?

To investigate the interaction between SCN4B and RhoA signaling pathways:

• Proximity Ligation Assay (PLA):

  • Use specific antibodies against SCN4B and RhoA

  • Apply PLA protocols to visualize and quantify molecular proximity (<40nm)

  • Studies have shown close association between SCN4B/β4 protein and RhoA in control cells, while no signal was observed in SCN4B-knockdown cells

• RhoA Activity Measurements:

  • Use GST-rhotekin pull-down assays to quantify active GTP-bound RhoA

  • Compare RhoA activity between:

    • Control cells

    • SCN4B-knockdown cells

    • SCN4B-overexpressing cells

  • Western blot analysis of pulled-down proteins indicates RhoA activity

• Downstream Effector Analysis:

  • Examine phosphorylation status of ROCK, LIMK, and cofilin

  • Quantify stress fiber formation through F-actin staining

  • Measure myosin light chain phosphorylation as an indicator of actomyosin contractility

• Pharmacological Interventions:

  • Use RhoA inhibitors (e.g., C3 transferase) or activators

  • Apply myosin II inhibitors (blebbistatin) to determine if they can rescue invasive phenotypes

  • Test ROCK inhibitors (Y-27632) to assess the involvement of the RhoA-ROCK pathway

• Live Cell Imaging:

  • Track cell morphology changes using phase-contrast microscopy

  • Measure cell circularity index as an indicator of amoeboid-mesenchymal transitions

  • Monitor real-time RhoA activity using FRET-based biosensors

These methodological approaches can help elucidate the molecular mechanisms by which SCN4B regulates RhoA activity and subsequent effects on cell migration, invasion, and cancer progression.

What are the emerging research areas involving SCN4B beyond its classical role in sodium channel function?

Research on SCN4B has expanded significantly beyond its classical role as an auxiliary subunit of voltage-gated sodium channels, opening several promising research areas:

• Cancer biology and metastasis suppression:

  • SCN4B functions as a metastasis-suppressor gene

  • Its reduced expression correlates with high-grade primary and metastatic tumors

  • It regulates cancer cell migration and invasiveness through RhoA signaling pathways

• Immunology and T-cell development:

  • SCN4B is essential for positive selection of CD4+ T cells

  • Its extracellular Ig domain regulates the SCN5a pore in cis

  • This reveals unexpected roles in immune system development

• Cell adhesion and extracellular matrix interactions:

  • SCN4B proteins can function as cell adhesion molecules (CAMs)

  • They contain immunoglobulin-like extracellular domains that mediate these interactions

  • This suggests roles in tissue architecture maintenance

• Amoeboid-mesenchymal transition regulation:

  • SCN4B influences cancer cell invasion modes

  • Reduced expression promotes acquisition of an amoeboid–mesenchymal hybrid phenotype

  • This advances our understanding of cellular plasticity in metastasis

These emerging areas highlight the multifunctional nature of SCN4B and suggest it may serve as a promising therapeutic target or biomarker in various pathological conditions beyond channelopathies.

How might SCN4B function as a potential therapeutic target, and what methodological approaches would be needed to develop targeted therapies?

SCN4B's potential as a therapeutic target stems from its diverse functions, particularly in cancer and immunology. Developing targeted therapies would require several methodological approaches:

• Structure-based drug design:

  • Determine the three-dimensional structure of SCN4B protein domains

  • Identify druggable pockets using computational modeling

  • Design small molecules that could mimic the function of specific domains

• Functional domain peptide mimetics:

  • Develop peptides based on the C-terminus of SCN4B, which has been shown to prevent hyperactivated migration

  • Create cell-penetrating peptides that could deliver functional domains intracellularly

  • Test these peptides in cancer cell models to verify their ability to restore normal cell behavior

• Gene therapy approaches:

  • Develop vectors for SCN4B overexpression in tumor cells

  • Research has shown that overexpression of SCN4B reduces cancer cell invasiveness and tumor progression

  • Design targeted delivery systems for tissue-specific expression

• RhoA pathway modulators:

  • Identify compounds that inhibit the increased RhoA activity seen with SCN4B reduction

  • Develop combination therapies targeting both SCN4B expression and RhoA signaling

  • Test in animal models of metastatic cancer

• Biomarker development:

  • Standardize immunohistochemical detection methods for SCN4B in clinical samples

  • Develop prognostic panels incorporating SCN4B expression levels

  • Correlate with clinical outcomes in large patient cohorts

Therapeutic development would need to address the tissue-specific functions of SCN4B to minimize off-target effects, particularly on cardiac and neuronal tissues where SCN4B functions as a sodium channel modulator.