SNTA1 Antibody

What is SNTA1 and where is it primarily expressed?

SNTA1 (syntrophin, alpha 1) is a cytoplasmic peripheral membrane scaffold protein that functions as a component of the dystrophin-associated protein complex. It serves as a signal-transducing adaptor protein and scaffold for various signaling molecules. Structurally, SNTA1 contains a PDZ domain, two Pleckstrin homology domains, and a 'syntrophin unique' domain that enable its diverse protein interactions. The protein shows high expression primarily in skeletal muscle and cardiac tissue, where it plays crucial roles in cellular signaling and structural integrity .

What is the molecular weight of SNTA1 protein?

SNTA1 has a calculated molecular weight of 54 kDa, corresponding to its 505 amino acid sequence. This observed molecular weight has been consistently confirmed in Western blot analyses across human, mouse, and rat samples. When running gel electrophoresis, researchers should expect to observe a distinct band at approximately 54 kDa when using validated SNTA1 antibodies under reducing conditions .

What are the primary functions of SNTA1 in cellular physiology?

SNTA1 functions as a crucial scaffolding protein that orchestrates signal transduction complexes by clustering various signaling components. It plays significant roles in:

• Neuromuscular junction development through its interaction with dystrophin

• Regulation of vasoconstriction by circulating norepinephrine

• Modulation of sodium channel function through direct interaction with Nav1.5

• Regulation of nitric oxide synthesis through its association with neuronal nitric oxide synthase (nNOS) and plasma membrane Ca²⁺/calmodulin-dependent ATPase (PMCA4b)

• Cell migration and reactive oxygen species (ROS) generation through its involvement in Rac1 activation

What are the validated applications for SNTA1 antibodies?

SNTA1 antibodies have been successfully validated for multiple applications in molecular and cellular research. Western blotting (WB) is the most commonly validated application, with optimal dilution ranges typically between 1:500-1:3000 depending on the specific antibody formulation and sample type. Immunohistochemistry (IHC) represents another validated application, with recommended dilutions of 1:50-1:500. Additionally, SNTA1 antibodies have been successfully employed in ELISA assays. For optimal results, it is recommended to titrate the antibody concentration for each specific experimental system and sample type .

How should I optimize Western blot protocols for SNTA1 detection?

For optimal Western blot detection of SNTA1:

• Sample preparation: Use RIPA or NP-40 based lysis buffers supplemented with protease inhibitors to extract total protein from tissues or cells.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for optimal separation around the 54 kDa range.

• Transfer: Semi-dry or wet transfer methods are suitable, with PVDF membranes generally preferred over nitrocellulose for SNTA1 detection.

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature.

• Primary antibody incubation: Apply SNTA1 antibody at dilutions between 1:500-1:3000 in blocking buffer, incubating overnight at 4°C.

• Detection: Use appropriate secondary antibodies conjugated to HRP, followed by ECL detection.

• Expected results: A specific band at approximately 54 kDa should be observed in heart and skeletal muscle tissues from human, mouse, and rat samples .

What tissue preparation methods are recommended for immunohistochemical detection of SNTA1?

For optimal immunohistochemical detection of SNTA1 in tissue sections:

• Fixation: 10% neutral buffered formalin fixation for 24-48 hours is recommended.

• Sectioning: 4-6 μm thickness paraffin sections are optimal for SNTA1 detection.

• Antigen retrieval: Two validated methods have been demonstrated:

  • Primary method: TE buffer pH 9.0 heat-induced epitope retrieval

  • Alternative method: Citrate buffer pH 6.0 heat-induced epitope retrieval

• Blocking: 3-5% normal serum (matching the species of the secondary antibody) in PBS or TBS for 1 hour at room temperature.

• Primary antibody: Apply SNTA1 antibody at dilutions between 1:50-1:500 in antibody diluent, incubating overnight at 4°C.

• Detection: Use appropriate detection systems (HRP/DAB or fluorescent-based) according to laboratory protocols.

• Positive control tissues: Heart tissue sections are recommended as positive controls for SNTA1 immunostaining .

How can SNTA1 antibodies be used to investigate protein-protein interactions in the dystrophin complex?

SNTA1 antibodies provide valuable tools for investigating protein-protein interactions within the dystrophin complex through several methodological approaches:

• Co-immunoprecipitation (Co-IP): SNTA1 antibodies can effectively immunoprecipitate SNTA1 and its associated protein complexes from tissue or cell lysates. This technique has successfully demonstrated interactions between SNTA1 and several key proteins, including dystrophin, nNOS, PMCA4b, and sodium channel Nav1.5. For optimal results, use mild lysis conditions (1% NP-40 or 0.5% Triton X-100) to preserve protein complexes.

• Proximity ligation assays (PLA): This technique allows visualization of protein interactions in situ, providing spatial information about SNTA1 interactions within cellular compartments, particularly at the sarcolemma and postsynaptic membrane.

• Pull-down assays: Using purified components and SNTA1 antibodies to verify direct protein interactions and map specific interaction domains.

Research has demonstrated that SNTA1 directly interacts with the PDZ domain-binding motif formed by the last three residues (serine-isoleucine-valine) of Nav1.5 C terminus, and is associated with the nNOS-PMCA4b complex in cardiomyocytes .

What role does SNTA1 play in cardiac sodium channel function and arrhythmias?

SNTA1 has been identified as a critical regulator of cardiac sodium channel (Nav1.5) function through complex molecular mechanisms:

• Direct interaction: SNTA1 directly associates with Nav1.5 through its PDZ domain, forming a macromolecular complex that regulates channel activity and membrane localization.

• Modulation of nitric oxide signaling: SNTA1 forms a regulatory complex with nNOS and PMCA4b that influences sodium channel function through nitric oxide-dependent mechanisms. Specifically, PMCA4b acts as a potent inhibitor of NO synthesis when in complex with SNTA1 and nNOS.

• Pathogenic implications: Mutations in SNTA1 (such as A390V and A261V) have been identified in patients with cardiac arrhythmias, particularly Long QT Syndrome (LQTS), designated as LQT12.

Experimental data shows that certain SNTA1 mutations can disrupt the association with PMCA4b and antagonize the inhibition of nNOS, resulting in augmentation of both peak and late sodium currents (INa). For example, co-expression of A261V-SNTA1 with wild-type SCN5A (encoding Nav1.5) significantly increased late sodium current to 0.44 ± 0.1% compared to 0.13 ± 0.03% with wild-type proteins, demonstrating a mechanistic link between SNTA1 dysfunction and arrhythmia pathogenesis .

*P < 0.05 vs. wild-type (WT)-SCN5A+WT-SNTA1

How does SNTA1 contribute to Rac1 activation and cancer cell migration?

SNTA1 plays a significant role in Rac1 activation through a novel molecular mechanism involving P66shc and Grb2 proteins:

• Complex formation: SNTA1 forms a complex with P66shc and Grb2 proteins, as demonstrated through co-immunoprecipitation assays.

• Displacement mechanism: When SNTA1 and P66shc are overexpressed in cancer cells, they cause significant displacement of Sos1 protein from Grb2. This results in Sos1 predominantly forming a complex with Eps8 and E3b1, which activates Rac1.

• Downstream effects: SNTA1/P66shc-mediated Rac1 activation leads to:

  • Increased reactive oxygen species (ROS) production

  • Enhanced migratory potential in human breast cancer cells

  • Acquisition of malignant phenotypes

This research reveals SNTA1 as a potential target for interventions aimed at modulating cancer cell migration and invasiveness. Experimental approaches using siRNAs and shRNAs to downregulate SNTA1 expression have effectively reduced Rac1 activation and subsequently decreased migration in breast cancer cell models .

What are common challenges in SNTA1 antibody Western blot experiments and how can they be overcome?

Several challenges may arise when performing Western blot analysis with SNTA1 antibodies:

• Cross-reactivity issues:

  • Problem: Some SNTA1 antibodies may cross-react with other syntrophin family members (β1, β2, γ1, γ2 syntrophins) due to sequence homology.

  • Solution: Validate antibody specificity using positive and negative controls. Consider using knockout/knockdown samples or comparing with other validated antibodies.

• Signal intensity variations:

  • Problem: Weak signals may be observed despite adequate protein loading.

  • Solution: Optimize antibody concentration (1:500-1:3000 range), extend primary antibody incubation time to overnight at 4°C, use high-sensitivity ECL substrates, and ensure proper antigen retrieval in sample preparation.

• Non-specific bands:

  • Problem: Multiple bands may appear at unexpected molecular weights.

  • Solution: Increase blocking time and concentration (5% milk or BSA for 2 hours), perform more stringent washing steps, and optimize antibody dilution.

• Sample degradation:

  • Problem: Degraded SNTA1 protein may result in multiple lower molecular weight bands.

  • Solution: Use fresh tissue/cell samples, include protease inhibitors in lysis buffers, maintain samples at cold temperatures during processing, and avoid repeated freeze-thaw cycles .

How should I select the appropriate positive and negative controls for SNTA1 antibody validation?

Proper control selection is critical for validating SNTA1 antibody specificity and performance:

Positive controls:

• Tissue samples:

  • Mouse heart tissue and rat heart tissue have been validated as reliable positive controls for SNTA1 detection in Western blot applications.

  • Mouse heart tissue is recommended for immunohistochemistry applications.

• Cell lines:

  • Cardiomyocyte cell lines (e.g., HL-1, H9c2)

  • Skeletal muscle cell lines (e.g., C2C12)

  • Cells transfected with SNTA1 expression constructs provide strong positive controls

Negative controls:

• SNTA1 knockout or knockdown samples:

  • Tissues from Snta1 knockout mice

  • Cells treated with validated siRNA or shRNA against SNTA1

• Non-expressing tissues/cells:

  • Certain cell lines with minimal SNTA1 expression (verify through literature)

  • SNTA1 antibody pre-absorbed with the immunizing peptide

• Technical controls:

  • Primary antibody omission

  • Isotype control antibodies

When validating a new SNTA1 antibody, researchers should perform side-by-side comparisons with previously validated antibodies and confirm specificity through multiple applications .

What are the key considerations for optimizing SNTA1 immunoprecipitation experiments?

Successful immunoprecipitation of SNTA1 and its associated proteins requires careful optimization:

• Lysis buffer selection:

  • Use mild lysis buffers (1% NP-40, 0.5% Triton X-100) to preserve protein-protein interactions

  • Include appropriate protease and phosphatase inhibitors

  • Consider specialized buffers for membrane protein extraction when studying SNTA1 interactions with membrane proteins like Nav1.5

• Antibody selection and coupling:

  • Choose antibodies raised against epitopes not involved in protein-protein interactions

  • Consider pre-coupling antibodies to protein A/G beads to reduce background

  • Optimal antibody amount typically ranges from 2-5 μg per immunoprecipitation reaction

• Incubation conditions:

  • Extend incubation time (overnight at 4°C) to enhance protein complex capture

  • Use gentle rotation rather than vigorous shaking to preserve complexes

• Washing stringency:

  • Adjust salt and detergent concentrations in wash buffers based on interaction strength

  • Perform 3-5 wash steps to remove non-specific binding while preserving specific interactions

• Elution methods:

  • For downstream mass spectrometry, consider native elution with peptide competition

  • For Western blot analysis, standard SDS elution buffers are suitable

• Confirming specificity:

  • Include IgG control immunoprecipitations

  • Validate interactions through reciprocal immunoprecipitations

These optimizations are essential when investigating SNTA1's interactions with key binding partners such as dystrophin, nNOS, PMCA4b, and Nav1.5 sodium channels .

How can I interpret SNTA1 expression patterns in disease models?

Interpreting SNTA1 expression patterns requires consideration of several factors:

• Tissue-specific expression baseline:

  • SNTA1 expression is normally highest in skeletal muscle and heart tissue

  • Lower expression levels occur in nervous system tissues

  • Minimal expression in most other tissues under normal conditions

• Subcellular localization:

  • In normal tissues: Primarily localized to the sarcolemma in muscle and postsynaptic membrane in neurons

  • In disease states: May show altered localization (cytoplasmic redistribution, nuclear accumulation, etc.)

• Disease-specific alterations:

  • Muscular dystrophies: SNTA1 expression and localization may be altered secondary to dystrophin complex disruption

  • Cardiac arrhythmias: Expression levels may remain normal, but functional interactions with cardiac ion channels may be disrupted

  • Cancer models: Potential upregulation in certain cancers, associated with increased cell migration and ROS production

• Analytical approaches:

  • Quantitative Western blotting with normalization to appropriate loading controls

  • Immunohistochemical scoring systems evaluating both intensity and distribution patterns

  • Correlation of expression with clinical parameters and outcomes in patient samples

When analyzing SNTA1 expression in disease contexts, comparing multiple methodologies (protein and mRNA levels, subcellular distribution) provides the most comprehensive assessment .

What genetic models are available for studying SNTA1 function in vivo?

Several genetic models have been developed to investigate SNTA1 function in vivo:

• Knockout mouse models:

  • Complete Snta1 knockout mice are viable and have been used to study the role of SNTA1 in various physiological processes

  • These models have revealed roles for SNTA1 in neuromuscular junction development and regulation of vasoconstriction

• Knock-in models:

  • Models harboring specific mutations identified in human diseases (e.g., Long QT Syndrome mutations)

  • Allow for the investigation of specific functional alterations rather than complete protein loss

• Conditional knockout models:

  • Tissue-specific deletion of Snta1 using Cre-loxP technology

  • Enables the distinction between primary and secondary effects of SNTA1 loss in specific tissues

• Transgenic overexpression models:

  • Overexpression of wild-type or mutant SNTA1 to study gain-of-function effects

  • Useful for investigating the effects of SNTA1 upregulation in cancer and other pathological contexts

• Zebrafish models:

  • Emerging models for studying SNTA1 function in development and disease

When designing studies using these models, researchers should consider the potential for compensation by other syntrophin family members (β1, β2, γ1, and γ2-syntrophins), which may partially mask phenotypes in knockout models .

How can SNTA1 antibodies be utilized in translational research on cardiac arrhythmias?

SNTA1 antibodies offer valuable tools for translational research in cardiac arrhythmias:

• Diagnostic applications:

  • Evaluation of SNTA1 expression and localization in cardiac tissue from arrhythmia patients

  • Assessment of SNTA1-Nav1.5 interactions in patient samples to identify potential functional disruptions

• Mechanistic investigations:

  • Immunoprecipitation to study the composition of macromolecular complexes containing SNTA1 and cardiac ion channels

  • Immunofluorescence to evaluate co-localization of SNTA1 with Nav1.5 and regulatory proteins at the intercalated disc

• Therapeutic target validation:

  • Antibody-based detection of changes in SNTA1-protein interactions following drug treatment

  • Evaluation of therapeutics designed to modulate SNTA1-dependent regulation of sodium channels

• Personalized medicine approaches:

  • Screening for SNTA1 mutations and corresponding protein expression/function in arrhythmia patients

  • Correlation of SNTA1 status with clinical outcomes and treatment responses

Research has established that mutations in SNTA1 (such as A390V and A261V) can disrupt normal regulation of cardiac sodium channels, leading to Long QT Syndrome (LQT12). Electrophysiological studies have demonstrated that these mutations can significantly increase late sodium current, providing a mechanistic explanation for the arrhythmogenic potential of SNTA1 dysfunction .