SCN8A Antibody

What is SCN8A and why is it significant in neurological research?

SCN8A encodes the voltage-gated sodium channel Nav1.6, a multi-pass transmembrane protein that mediates sodium ion permeability of excitable membranes. This channel is critical for rapid signal transduction and plays a significant role in neuronal development. The protein consists of a pore-forming alpha subunit, a noncovalently associated beta1 subunit, and a disulfide-linked beta2 subunit. These beta subunits regulate channel expression levels, modulate gating, and function as cell adhesion molecules for cellular aggregation and cytoskeleton interaction .

The significance of SCN8A in neurological research stems from its crucial role in neuronal function and its association with various neurological disorders. Loss of SCN8A expression can result in progressive paralysis and early death, underscoring its biological importance . Furthermore, genetic variants in SCN8A underlie a wide spectrum of neurodevelopmental phenotypes ranging from severe epileptic encephalopathy to benign familial infantile epilepsy to neurodevelopmental delays with or without seizures . Understanding SCN8A function through antibody-based detection methods provides critical insights into neuronal physiology and pathophysiology.

What types of SCN8A antibodies are available and what are their key differences?

Several types of SCN8A antibodies are available for research purposes, each with distinct characteristics:

Polyclonal antibodies recognize multiple epitopes on the SCN8A protein, potentially providing higher sensitivity but sometimes with lower specificity. These antibodies are often preferred for detecting low levels of native proteins in complex samples. The PA5-37276 antibody from Thermo Fisher, for example, has >95% purity by SDS-PAGE and detects endogenous protein at 225 kDa .

Monoclonal antibodies like EPR25137-45 from Abcam recognize a single epitope, providing higher specificity and better lot-to-lot consistency . These characteristics make monoclonal antibodies particularly valuable for applications requiring precise quantification or when cross-reactivity must be minimized.

What applications are SCN8A antibodies validated for?

SCN8A antibodies have been validated for multiple experimental applications:

• Western Blot (WB): For detecting and quantifying SCN8A protein in cell or tissue lysates. Most antibodies detect full-length SCN8A at approximately 225 kDa .

• Immunohistochemistry (IHC): Both on paraffin-embedded (IHC-P) and frozen sections (IHC-Fr) for visualizing SCN8A distribution in tissue samples .

• Immunocytochemistry (ICC): For subcellular localization studies in cultured cells. For instance, the AB5580 antibody has been validated for ICC on rat dorsal root ganglion primary cultures at a 1:100 dilution .

• Immunoprecipitation (IP): For isolating SCN8A protein complexes and studying protein-protein interactions .

When selecting an SCN8A antibody, researchers should verify the validated applications for their specific antibody of interest and optimize protocols accordingly. For example, dilutions for immunocytochemistry should be made using a carrier protein such as BSA (1-3%) as recommended for some antibodies .

How can SCN8A antibodies be used to study splice isoform expression?

SCN8A exists in multiple splice isoforms that may have distinct functional properties. Of particular importance are the alternative splice variants involving exon 5, which can include either exon 5N or exon 5A. The National Center for Biotechnology Information (NCBI) designates the sequence with exon 5N as variant 1 (NM_014191) and the sequence with exon 5A as variant 3 (NM_001330260) .

When using antibodies to study these isoforms, researchers should consider:

• Epitope location relative to splice sites: Determine whether your antibody's epitope spans or includes regions affected by alternative splicing. Some antibodies may preferentially detect certain isoforms based on epitope location.

• Western blot analysis: Carefully analyze banding patterns, as different isoforms may have subtle size differences. High-resolution gels may be necessary to distinguish closely related variants.

• Validation in appropriate model systems: Use cellular or animal models with known expression of specific isoforms. The search results indicate that some studies compare functional properties of variants in different isoforms generated by alternative splicing of exon 5 .

• Complementary techniques: Combine antibody detection with RT-PCR or RNA sequencing to correlate protein expression with transcript levels of specific splice variants.

Research has demonstrated that many disease-associated SCN8A variants exhibit isoform-dependent functional effects , highlighting the importance of considering splice variants when interpreting antibody results.

What considerations are important when using SCN8A antibodies in epilepsy research?

SCN8A has been implicated in various forms of epilepsy, and antibodies can be valuable tools for studying its role in epileptogenesis and seizure mechanisms. Key considerations include:

When designing epilepsy-related studies with SCN8A antibodies, researchers should carefully select antibodies validated for the specific applications and tissue types relevant to their research question.

How can SCN8A antibodies contribute to genotype-phenotype correlation studies?

The International SCN8A Patient Registry collects comprehensive data from individuals with SCN8A variants to better understand genotype-phenotype correlations . Antibodies can significantly enhance these efforts by providing molecular insights:

• Expression analysis in model systems: Use antibodies to determine how different variants affect SCN8A protein expression, stability, or subcellular localization in cellular or animal models.

• Functional domain mapping: Compare antibody binding patterns between wild-type and variant SCN8A to assess potential structural changes that might explain functional differences.

• Patient-derived samples: When available and ethical, analyze SCN8A expression in patient-derived samples (e.g., fibroblasts, induced neurons) using validated antibodies.

• Correlation with clinical data: Link antibody-based molecular findings with clinical information from patient registries. For example, researchers might investigate whether variants that reduce protein expression (detectable by antibodies) correlate with specific phenotypic features.

The International SCN8A Patient Registry has been particularly valuable for establishing associations between genetic variants and clinical presentations . Antibody-based studies can complement registry data by providing mechanistic insights into how different variants affect protein function and expression.

What are optimal sample preparation methods for detecting SCN8A in different experimental contexts?

SCN8A is a large (225 kDa), membrane-bound protein that requires careful sample preparation for optimal detection:

For Western Blot analysis:

• Use strong lysis buffers containing appropriate detergents to solubilize membrane proteins effectively

• Include protease inhibitors to prevent degradation

• Avoid excessive heating of samples to prevent aggregation

• Use lower percentage gels (6-8%) to adequately resolve high molecular weight proteins

• Consider longer transfer times or specialized transfer systems for efficient transfer of large proteins

For Immunohistochemistry:

• Optimize fixation conditions (paraformaldehyde concentration and duration)

• Consider antigen retrieval methods to expose epitopes potentially masked during fixation

• Ensure adequate tissue permeabilization while maintaining morphology

• Block endogenous peroxidase activity if using HRP-based detection systems

For Immunocytochemistry:

• The Sigma-Aldrich AB5580 antibody has been successfully used on rat dorsal root ganglion primary cultures at a dilution of 1:100 , which can serve as a starting point

• Dilutions should be made using a carrier protein such as BSA (1-3%)

• Optimize fixation and permeabilization protocols for your specific cell type

How can antibody specificity be validated for SCN8A research?

Thorough validation is crucial for ensuring reliable results with SCN8A antibodies:

• Genetic validation:

  • Testing on tissues from SCN8A knockout or knockdown models

  • Comparing signal in cells with endogenous versus overexpressed SCN8A

  • Evaluating specificity in heterologous expression systems

• Biochemical validation:

  • Confirming detection of the expected molecular weight band (~225 kDa for full-length SCN8A)

  • Performing pre-absorption controls with immunizing peptides when available

  • Conducting immunoprecipitation followed by mass spectrometry analysis

• Cross-validation with multiple antibodies:

  • Using antibodies targeting different epitopes to confirm findings

  • Comparing results from both monoclonal and polyclonal antibodies

• Negative controls:

  • Testing in cells known not to express SCN8A

  • The search results mention that researchers did not detect Nav1.6 protein in ND7/23 cell lysates using two different polyclonal antibodies validated against mouse brain , demonstrating the importance of appropriate negative controls

Proper validation enhances confidence in experimental results and helps distinguish true biological findings from technical artifacts.

What factors can affect SCN8A antibody performance in electrophysiological studies?

When combining antibody-based detection with electrophysiological studies of SCN8A:

• Antibody access to the target:

  • The multi-pass transmembrane nature of SCN8A means that some epitopes may be inaccessible in intact cells

  • Consider the location of the epitope (extracellular, transmembrane, or intracellular) when designing experiments

• Functional interference:

  • Antibody binding might directly affect channel function

  • For functional studies, consider using separate samples for electrophysiology and antibody detection

• Cell viability and membrane integrity:

  • Fixation procedures for antibody detection may compromise electrophysiological properties

  • Plan experimental sequences carefully (typically electrophysiology first, then antibody studies)

• Expression systems:

  • Different expression systems may yield varying results

  • The search results describe using ND7/23 cells for electrophysiological recordings of SCN8A variants together with human β1-and β2-subunits of voltage-gated Na+ channels

• Data correlation approaches:

  • Consider single-cell approaches that allow correlation between function and expression

  • Use batch analyses to relate average electrophysiological properties to protein expression levels

How should western blot data be interpreted when studying SCN8A expression levels?

When analyzing SCN8A western blot data:

• Molecular weight considerations:

  • Full-length SCN8A should be detected at approximately 225 kDa

  • Be alert for potential degradation products or processing fragments

  • Post-translational modifications can affect migration patterns

• Quantification approaches:

  • Normalize SCN8A signals to appropriate loading controls

  • For membrane proteins, consider normalizing to membrane-specific markers

  • Use densitometry software for quantitative analysis

  • Report relative expression changes rather than absolute values

• Statistical analysis:

  • Perform sufficient biological replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Consider power analysis to ensure adequate sample size

• Technical considerations:

  • High molecular weight proteins may transfer inefficiently

  • Signal saturation can lead to inaccurate quantification

  • Membrane proteins can form aggregates that don't enter the gel properly

How can conflicting SCN8A antibody results be reconciled?

When faced with conflicting results from different SCN8A antibodies:

• Evaluate antibody characteristics:

  • Different epitopes might be differentially accessible in various contexts

  • Polyclonal antibodies may detect epitopes masked or altered in certain sample preparations

  • Consider the validation history of each antibody

• Consider biological and technical factors:

  • Different splice isoforms might be preferentially detected by different antibodies

  • The search results note that disease-associated SCN8A variants can exhibit isoform-dependent functional effects

  • Post-translational modifications could mask epitopes

  • Sample preparation methods might affect epitope accessibility

• Validation approaches:

  • Use multiple antibodies targeting different epitopes

  • Correlate antibody results with mRNA expression data

  • Employ genetic approaches (knockdown/knockout) to confirm specificity

  • Consider orthogonal protein detection methods

• Reconciliation strategies:

  • Report all results transparently, including discrepancies

  • Investigate potential biological explanations for differences

  • Determine which antibody has undergone more rigorous validation

  • Use complementary techniques to resolve contradictions

How might SCN8A antibodies contribute to understanding ataxia phenotypes?

Recent research has established episodic or predominant chronic ataxia as syndromes caused by de novo and familial missense variants or haploinsufficiency in SCN8A . Antibodies can contribute to understanding these phenotypes through:

• Expression pattern analysis: Investigating whether ataxia-associated variants affect SCN8A expression levels or distribution in cerebellar tissues and other relevant brain regions.

• Mechanistic studies: Combining antibody detection with electrophysiological recordings to correlate protein expression with functional alterations in ataxia models.

• Cellular model validation: Using antibodies to verify expression of ataxia-associated variants in heterologous expression systems for functional studies.

• Therapeutic target validation: Evaluating potential therapeutics targeting SCN8A expression or function in ataxia models.

The search results describe research where ataxia-related SCN8A variants were engineered into TTX-resistant Nav1.6 channel constructs for functional studies . Antibody-based detection can complement such approaches by confirming expression and localization of these variants.

What are the future prospects for SCN8A antibodies in precision medicine approaches?

SCN8A antibodies could support precision medicine approaches for SCN8A-related disorders:

• Patient stratification:

  • Identifying molecular phenotypes that predict treatment response

  • Distinguishing between gain-of-function and loss-of-function effects at the protein level

• Therapeutic monitoring:

  • Assessing changes in SCN8A expression or localization after therapeutic intervention

  • Determining target engagement for SCN8A-directed therapies

• Biomarker development:

  • Correlating protein expression patterns with disease severity or progression

  • Identifying signatures that predict response to sodium channel modulators

• Companion diagnostics:

  • Supporting development of diagnostic tools to guide treatment selection

  • Enabling monitoring of treatment efficacy

The International SCN8A Patient Registry notes that "Registry data will play a key role in assisting with establishment of the first clinical treatment guidelines for SCN8A-related disorders" . Antibody-based research could complement these efforts by providing molecular insights to guide personalized treatment approaches.