SCN7A Antibody

What is SCN7A and what is its functional significance in biological systems?

SCN7A, also known as Nax, Nav2.1, or Nav2.3, is an atypical member of the SCNA family of voltage-dependent sodium channels. Unlike typical voltage-gated sodium channels, SCN7A functions as a sodium leak channel that:

• Acts as an osmosensor regulating sodium ion levels in various tissues and organs

• Mediates sodium influx through membranes along concentration gradients rather than voltage gates

• Contains 4 internal repeats, each with 5 hydrophobic segments (S1, S2, S3, S5, S6) and one positively charged segment (S4)

Key distinguishing features include:

• Lower sequence similarity between human and mouse proteins compared to other orthologous sodium channel pairs

• Fewer arginine and lysine residues in the S4 segments (which typically sense voltage changes)

• Functions in sensing body-fluid sodium levels and controlling salt intake behavior

The reported molecular weights for SCN7A protein are:

• Calculated molecular weight: 193 kDa

• Observed molecular weight: 193-260 kDa (variation likely due to post-translational modifications)

This difference between calculated and observed weights is important to consider when interpreting Western blot results. The higher observed weight may be attributed to glycosylation and other post-translational modifications typical of membrane proteins .

In which tissues is SCN7A protein most highly expressed?

SCN7A shows tissue-specific expression patterns that researchers should consider when selecting positive controls:

Immunohistochemical studies have specifically detected SCN7A in mouse heart tissue and subfornical organ (SFO) glial cells, which are important regions for sodium sensing .

What are the recommended protocols for Western blot detection of SCN7A?

For optimal Western blot detection of SCN7A:

• Sample preparation:

  • Use mouse skeletal muscle tissue, mouse/rat heart tissue, or human samples expressing endogenous SCN7A

  • Prepare tissue lysates using standard RIPA buffer with protease inhibitors

• Electrophoresis conditions:

  • Use 6-8% SDS-PAGE gels (recommended for large proteins >150 kDa)

  • Run at lower voltage (80-100V) to improve resolution of high molecular weight proteins

• Transfer parameters:

  • Wet transfer is recommended for large proteins

  • Transfer at 30V overnight at 4°C using 0.05% SDS in transfer buffer

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:1000

  • Incubate overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG (1:5000)

• Detection:

  • Use high-sensitivity ECL substrates suitable for large, potentially low-abundance proteins

  • Observed molecular weight should be approximately 193-260 kDa

What are the best practices for immunohistochemistry detection of SCN7A?

For immunohistochemical detection of SCN7A:

• Tissue preparation:

  • Perfusion-fixed frozen sections yield optimal results for brain tissue

  • For heart tissue, antigen retrieval with TE buffer pH 9.0 is suggested

  • Alternative: citrate buffer pH 6.0 for antigen retrieval

• Antibody dilution and incubation:

  • Primary antibody: 1:50-1:500 dilution range

  • For fluorescent detection: Use goat anti-rabbit-AlexaFluor-488 as secondary antibody (1:300-1:500)

  • Include DAPI counterstain for nuclear visualization

• Specificity controls:

  • Negative control: Pre-incubation of antibody with SCN7A blocking peptide

  • Positive control tissues: mouse heart tissue, subfornical organ

• Special considerations:

  • Expression in glial profiles may require co-staining with glial markers

  • For ventromedial hypothalamus (VMH) and subfornical organ (SFO), specific anatomical marking is important

How should SCN7A antibodies be stored to maintain activity?

Proper storage of SCN7A antibodies is crucial for maintaining their activity:

• Long-term storage:

  • Store at -20°C (most manufacturers recommend this temperature)

  • Antibodies are typically stable for one year after shipment under these conditions

• Storage buffer composition:

  • Most SCN7A antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some preparations may contain 0.1% BSA for additional stability

• Handling recommendations:

  • Avoid repeated freeze-thaw cycles

  • For antibodies stored at -20°C, aliquoting is generally unnecessary when in 50% glycerol

  • Allow antibody to equilibrate to room temperature before opening

• Working dilution stability:

  • Diluted antibody solutions should be prepared fresh

  • Store working dilutions at 4°C and use within 24-48 hours

What is the significance of SCN7A in cancer research, particularly in gastric cancer?

SCN7A has emerged as an important gene in cancer research, with particular relevance to gastric cancer:

These findings suggest SCN7A may serve as a potential biomarker for gastric cancer prognosis and could be valuable for patient stratification in clinical research.

How can I validate the specificity of SCN7A antibodies in my experimental system?

Rigorous validation of SCN7A antibody specificity is essential for reliable research outcomes:

• Blocking peptide experiments:

  • Pre-incubate the SCN7A antibody with its specific blocking peptide

  • Perform parallel experiments (Western blot, IHC, IF) with and without peptide pre-incubation

  • Specific signals should be eliminated or significantly reduced after peptide blocking

• Validation across multiple applications:

  • Test the antibody in at least two independent techniques (e.g., WB and IHC)

  • Ensure consistent molecular weight detection in Western blot (193-260 kDa)

  • Verify tissue expression patterns match known SCN7A distribution

• Knockout/knockdown controls:

  • Use SCN7A knockout tissues where available

  • Alternatively, use siRNA or shRNA knockdown samples as negative controls

  • Compare with wild-type or scrambled control samples

• Cross-species reactivity assessment:

  • Test the antibody on samples from multiple species (human, mouse, rat) when applicable

  • Be aware that there is lower sequence similarity between human and mouse SCN7A compared to other sodium channels

• Epitope mapping:

  • Verify the immunogen sequence used for antibody generation

  • Different antibodies target different regions of SCN7A:

    • Amino acids 265-368 (human SCN7A)

    • Amino acids 771-820 (human SCN7A)

    • Amino acids 842-856 (mouse SCN7A, intracellular 6th loop)

What are the key differences between SCN7A and other sodium channel proteins in experimental detection?

When studying SCN7A, it's crucial to distinguish it from other sodium channel family members:

• Structural and functional distinctions:

  • SCN7A is not voltage-gated, unlike other sodium channels in the family

  • Functions as a sodium leak channel that senses extracellular sodium concentration

  • Contains fewer positive charged residues in S4 segments compared to typical voltage-gated sodium channels

• Antibody cross-reactivity considerations:

  • Choose antibodies targeting unique regions of SCN7A to minimize cross-reactivity

  • SCN7A-specific antibodies typically target regions with low sequence homology to other sodium channels

  • Validate antibody specificity using tissues known to express SCN7A but not other sodium channels

• Functional assay distinctions:

  • SCN7A does not generate typical voltage-gated sodium currents

  • Functions as a sodium leak channel responding to extracellular sodium concentration changes

  • Its activity is regulated by endothelin signaling and associated with lactate production in glial cells

• Expression pattern differences:

  • SCN7A is prominently expressed in glial cells of specific brain regions, heart, and skin

  • Other sodium channels typically show neuronal or muscle expression patterns

  • Co-staining with cell-type specific markers can help distinguish SCN7A-expressing cells

How does SCN7A function as a sodium sensor in physiological systems?

SCN7A serves as an atypical sodium sensor with unique physiological roles:

• Sodium sensing mechanism:

  • Functions as a sodium leak channel rather than a voltage-gated channel

  • Allows sodium to flow through the membrane along its concentration gradient

  • Senses extracellular sodium levels particularly in central nervous system glial cells

• Physiological roles:

  • Controls salt intake behavior and voluntary water intake through activation of nearby neurons

  • Maintains appropriate sodium levels in the body

  • Mediates sodium influx into keratinocytes, playing a role in skin barrier homeostasis

• Regulation mechanisms:

  • Activity is regulated by endothelin signaling

  • Associated with lactate production in glial cells

  • May affect the digestion and absorption function of gastric epithelial cells

• Pathophysiological implications:

  • Upregulation occurs in scars and skin inflammation

  • Can activate the major sodium channel ENaC and downstream inflammatory signals

  • Regulates pathways like PAR-2 to promote release of inflammatory factors IL-1β and IL-8

  • Associated with tumor microenvironment in gastric cancer

What experimental systems are most suitable for studying SCN7A function?

Based on current research, several experimental systems are particularly effective for studying SCN7A:

• Cell culture models:

  • Glial cells from subfornical organ (SFO) for sodium sensing studies

  • Keratinocytes for skin barrier function research

  • Gastric cancer cell lines for tumor microenvironment studies

• Animal models:

  • SCN7A knockout mice (SCN7A−/−) to study behavioral responses to salt intake

  • Mouse models for studying SCN7A expression in heart tissue and brain regions

  • Rat models for studying SCN7A in subfornical organ and hippocampus

• Tissue analysis approaches:

  • Immunohistochemistry of brain sections focusing on subfornical organ and ventromedial hypothalamus

  • Western blot analysis of heart and skeletal muscle tissues

  • Co-immunostaining with glial markers for brain region studies

• Functional assays:

  • Sodium flux measurements to assess channel activity

  • Behavioral tests for salt preference in animal models

  • Gene expression analysis in cancer tissues with correlation to clinical outcomes

• Bioinformatic approaches:

  • GSEA analysis to identify enriched pathways associated with SCN7A expression

  • CIBERSORT algorithm to calculate immune cell infiltration in relation to SCN7A expression

  • Correlation analysis between SCN7A expression and clinical characteristics

What are the common challenges in detecting SCN7A protein and how can they be addressed?

Researchers frequently encounter several challenges when working with SCN7A antibodies:

• High molecular weight detection issues:

  • Problem: Inefficient transfer of large proteins (193-260 kDa)

  • Solution: Use wet transfer systems with 0.05% SDS in transfer buffer, transfer overnight at low voltage (30V) at 4°C

• Low signal intensity:

  • Problem: Low endogenous expression levels in some tissues

  • Solutions:

    • Increase protein loading (50-100 μg total protein)

    • Use high-sensitivity ECL detection systems

    • Extend primary antibody incubation (overnight at 4°C)

    • Optimize antibody concentration with titration experiments

• Non-specific binding:

  • Problem: Additional bands in Western blot

  • Solutions:

    • Increase blocking duration (2 hours or overnight)

    • Use 5% BSA instead of milk for blocking

    • Include 0.1% Tween-20 in wash buffers

    • Validate with blocking peptide experiments

• Antigen masking in fixed tissues:

  • Problem: Poor signal in IHC applications

  • Solutions:

    • Test different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

    • Optimize fixation protocols (shorter fixation times)

    • Try frozen sections instead of paraffin-embedded tissues

• Sample degradation:

  • Problem: Inconsistent results between experiments

  • Solutions:

    • Use fresh samples when possible

    • Add protease inhibitors to extraction buffers

    • Avoid repeated freeze-thaw cycles of samples

How do I select the most appropriate SCN7A antibody for my specific research application?

Selecting the optimal SCN7A antibody requires consideration of several factors:

• Experimental application requirements:

  • Western blot: Choose antibodies validated for WB with documented molecular weight detection

  • IHC: Select antibodies specifically validated for IHC with demonstrated tissue specificity

  • Multiple applications: Consider antibodies validated across several techniques

• Epitope considerations:

  • Different regions of SCN7A are targeted by various antibodies:

    • N-terminal region (amino acids 265-368)

    • Middle region (amino acids 771-820)

    • C-terminal region/intracellular loops (amino acids 842-856)

  • Access to the epitope may vary between applications (denatured vs. native conditions)

• Species reactivity requirements:

  • Human studies: Multiple antibodies available with validated human reactivity

  • Mouse studies: Select antibodies specifically validated in mouse tissues

  • Cross-species studies: Choose antibodies with documented reactivity across your species of interest

• Validation data availability:

  • Prioritize antibodies with extensive validation data in your application of interest

  • Look for blocking peptide controls, knockout validation, or multiple detection methods

  • Consider antibodies with published literature citations

• Clonality considerations:

  • Polyclonal antibodies: May provide stronger signals but potentially more background

  • Monoclonal antibodies: Typically more specific but may be sensitive to epitope masking

The table below summarizes key attributes of selected SCN7A antibodies from the search results: