selenon Antibody

What is SELENON and why is it important in research applications?

SELENON (Selenoprotein N, previously known as SEPN1) is a glycoprotein localized in the endoplasmic reticulum (ER) membrane with a molecular weight of approximately 65.8 kDa and 590 amino acid residues . It plays a crucial role in cell protection against oxidative stress and regulation of redox-related calcium homeostasis. SELENON modulates ryanodine receptor (RyR) activity by:

• Protecting RyR from oxidation under increased oxidative stress

• Directly controlling RyR redox state

• Regulating RyR-mediated calcium mobilization needed for normal muscle development and differentiation

Within the ER, SELENON reduces cysteinyl sulfenic acid formation in the calcium pump ATP2A2 back to free thiol, restoring its activity against oxidoreductase ERO1A-mediated oxidative damage . This protein is widely expressed in multiple tissues including skeletal muscle, brain, lung, and placenta, making it relevant for research across multiple physiological systems .

Mutations in the SELENON gene result in SELENON-related myopathies (SELENON-RM), a group of early-onset muscle disorders characterized by axial weakness, rigid spine, and respiratory insufficiency . These clinical manifestations make SELENON antibodies invaluable tools for investigating the pathophysiology of these rare conditions.

What experimental applications are commonly used with SELENON antibodies?

SELENON antibodies are utilized in multiple experimental applications with varying degrees of optimization required:

When designing experiments, researchers should consider that successful detection often requires optimization of sample preparation methods that preserve the native conformation of SELENON while maintaining its antigenicity .

How do I select the appropriate SELENON antibody for my specific research needs?

Selection of SELENON antibodies should be guided by several critical factors:

• Target epitope location: Antibodies targeting different regions (N-terminal, C-terminal, or specific domains like the first histidine-rich region) may yield different results. Some commercially available antibodies target:

  • C-terminal region (amino acids 450 to C-terminus)

  • Region adjacent to the first histidine-rich region (FHR)

  • Specific amino acid regions (e.g., AA 135-219)

• Species reactivity: Confirm cross-reactivity with your study species. Many antibodies react with:

  • Human SELENON

  • Mouse selenon

  • Rat selenon

  • Additional species (bovine, pig, zebrafish)

• Clonality:

  • Polyclonal antibodies: Offer broad epitope recognition but potential batch variation

  • Monoclonal antibodies: Provide consistent specificity but potentially narrower epitope recognition

• Validation data: Prioritize antibodies with:

  • Published citation records

  • Validation in multiple applications

  • Clear documentation of positive and negative controls

• Post-translational modifications: Consider whether glycosylation or other modifications at the epitope might affect antibody binding

How can SELENON antibodies contribute to investigating SELENON-related myopathies?

SELENON antibodies serve as critical tools for investigating SELENON-related myopathies (SELENON-RM) through multiple approaches:

• Diagnostic applications: SELENON antibodies can be used to assess protein expression in patient muscle biopsies, potentially complementing genetic testing. Reduced or aberrant SELENON expression patterns help distinguish SELENON-RM from other congenital myopathies with similar clinical presentations .

• Pathophysiological mechanisms: SELENON antibodies enable researchers to:

  • Characterize oxidation states of SELENON interaction partners like RyR and ATP2A2

  • Analyze calcium dynamics in affected tissues

  • Investigate mitochondrial integrity, which has recently been linked to SELENON function

• Therapeutic development: SELENON-neutralizing antibodies have potential therapeutic applications by:

  • Targeting specific functional domains to modulate SELENON activity

  • Serving as delivery vehicles for targeted drug approaches

  • Providing frameworks for designing selenomab-drug conjugates for precision therapy

• Biomarker discovery: Recent research has identified potential biomarkers through co-expression network analysis with SELENON. Antibody-based detection methods can validate these findings and establish reliable markers for disease progression and therapeutic response .

• Methodology for patient-derived models: When working with patient-derived cells or tissues, researchers should:

  • Use multiple antibodies targeting different epitopes to confirm findings

  • Include appropriate controls (healthy tissue, other muscular dystrophies)

  • Correlate protein expression with functional assays of oxidative stress and calcium homeostasis

What technical challenges exist in detecting SELENON in tissue and cellular samples?

Researchers face several technical challenges when detecting SELENON in biological samples:

• Subcellular localization: As an ER-resident transmembrane protein, SELENON detection requires:

  • Effective membrane permeabilization protocols

  • Preservation of ER structure during sample preparation

  • Careful selection of detergents for extraction that maintain epitope integrity

• Expression level variations: SELENON expression varies significantly across tissues and developmental stages:

  • Highest in skeletal muscle but with temporal regulation during development

  • Alternative splicing generates multiple isoforms that may not be detected by all antibodies

  • Post-transcriptional regulation through A-to-I RNA editing affects expression levels

• Fixation considerations: For immunohistochemistry/immunocytochemistry:

  • PFA fixation (4%) with Triton X-100 permeabilization has been successfully employed

  • Overfixation can mask epitopes through excessive protein crosslinking

  • Antigen retrieval methods must be optimized specifically for SELENON detection

• Co-detection challenges: When performing co-localization studies:

  • Use fluorophore combinations with minimal spectral overlap

  • Account for potential antibody cross-reactivity in multiplex assays

  • Consider sequential rather than simultaneous antibody incubations for better results

• Practical validation approach:

  • Employ positive controls of tissues known to express SELENON (skeletal muscle)

  • Include negative controls with SELENON-deficient samples or competing peptides

  • Confirm specificity through genetic knockdown/knockout validation when possible

How can selenomab technology improve SELENON-targeted therapeutics?

Selenomab technology represents an advanced approach to antibody engineering with significant implications for SELENON research and therapeutics:

• Fundamental principles of selenomab technology:

  • Selenomabs are engineered monoclonal antibodies containing one or more translatioinally incorporated selenocysteine (Sec) residues

  • The selenol group of selenocysteine provides unique chemical reactivity that allows for site-specific conjugation

  • This technology enables the production of homogeneous antibody-drug conjugates (ADCs) with precise drug-to-antibody ratios

• Advantages over conventional approaches:

  • Selenocysteine is particularly reactive compared to other amino acids, permitting fast, single-step, and efficient reactions under near physiological conditions

  • Selenomab-drug conjugates demonstrate excellent stability in human plasma and in circulation

  • Site-specific conjugation results in homogeneous products with improved therapeutic index compared to heterogeneous ADCs

• SELENON-specific applications:

  • Selenomabs could be engineered to target specific domains of SELENON for research purposes

  • In therapeutic contexts, anti-SELENON selenomabs could deliver targeted drugs to affected tissues

  • For SELENON-RM conditions, selenomab-based therapies might deliver compounds that compensate for SELENON deficiency

• Technical implementation considerations:

  • Current challenges include inefficient selenocysteine incorporation (yields of 2-4 mg/L compared to 20 mg/L for conventional antibodies)

  • Optimization strategies include using different SECIS elements (e.g., AUGA mutant of Toxoplasma gondii Selenoprotein T 3′UTR) and co-expression with SECIS binding protein 2 (SECISBP2)

  • Precise positioning of selenocysteine residues affects conjugation efficiency and final drug-to-antibody ratio

• Therapeutic potential:

  • Early research demonstrates selenomab-drug conjugates show potent and selective activity in diverse disease models

  • The technology could potentially be applied to deliver compounds targeting oxidative stress pathways relevant to SELENON function

  • Future development could include selenomabs carrying ERO1A inhibitors, which have shown promise in treating SELENON-RM in preclinical models

What methodologies exist for validating SELENON antibody specificity?

Rigorous validation of SELENON antibodies is essential for reliable research outcomes. Several complementary approaches should be employed:

• Genetic validation strategies:

  • siRNA/shRNA knockdown of SELENON in cell models to confirm signal reduction

  • CRISPR/Cas9 knockout models as negative controls

  • Comparison of antibody performance in tissues from wild-type vs. SELENON-mutant models

• Biochemical validation approaches:

  • Western blot analysis confirming the expected molecular weight (~65.8 kDa)

  • Antibody blocking with immunizing peptide to demonstrate specificity

  • Correlation of results across multiple antibodies targeting different SELENON epitopes

• Cross-application validation:

  • Correlation of results between immunohistochemistry, western blot, and immunofluorescence

  • Comparison of native vs. denatured protein detection to identify conformation-specific antibodies

  • Testing in multiple cell lines/tissues with known SELENON expression profiles

• Mass spectrometry validation:

  • Immunoprecipitation followed by mass spectrometry to confirm identity

  • Comparison of detected peptides with theoretical SELENON sequence

  • Analysis of post-translational modifications that might affect antibody binding

• Documentation and experimental design for validation:

How can SELENON antibodies be used to investigate the role of oxidative stress in muscle pathologies?

SELENON antibodies are valuable tools for investigating oxidative stress pathways in muscle disorders through several methodological approaches:

• Co-immunoprecipitation studies:

  • SELENON antibodies can be used to isolate protein complexes containing SELENON

  • This approach reveals interaction partners like ATP2A2 and ryanodine receptors

  • Changes in these interactions under oxidative stress conditions can be quantified

• Redox state analysis:

  • SELENON antibodies can be combined with redox-sensitive dyes or probes

  • This allows correlation between SELENON localization and cellular redox environment

  • Mutations affecting SELENON's redox function can be characterized by comparing wild-type and mutant protein interactions

• Calcium dynamics investigation:

  • By combining SELENON immunostaining with calcium imaging techniques

  • Researchers can correlate SELENON expression/localization with calcium flux

  • This approach has revealed SELENON's role in protecting the calcium pump ATP2A2 against oxidative damage

• Analysis of oxidative stress response pathways:

  • SELENON antibodies used in conjunction with antibodies against stress-response proteins

  • This reveals coordination between SELENON and broader cellular stress responses

  • Recent research identified ERO1A as a counterbalancing partner for SELENON, with therapeutic implications

• Oxidative damage markers correlation:

  • SELENON immunostaining combined with markers of protein oxidation, lipid peroxidation, or DNA damage

  • This approach quantifies relationships between SELENON dysfunction and cellular damage

  • Studies have shown SELENON-deficient tissues exhibit increased oxidative damage markers

Methodological recommendation: When investigating oxidative stress pathways, researchers should consider using approaches that preserve the native redox environment during sample preparation, as harsh extraction methods may disrupt the physiological redox state of SELENON and its partners.

What therapeutic approaches using SELENON antibodies are being investigated for SELENON-related myopathies?

Current research on SELENON antibody-based therapeutics for SELENON-RM is exploring several promising avenues:

• Neutralizing antibodies targeting disease mechanisms:

  • Similar to the approach used with selenoprotein P (SeP) neutralizing antibodies that improved glucose intolerance

  • These antibodies could potentially modulate pathological interactions without affecting beneficial functions

• Antibodies as biomarker tools:

  • Research teams are optimizing high-throughput drug screening approaches using SELENON antibodies to detect biomarker changes

  • This enables screening of FDA-approved drugs for repurposing in SELENON-RM treatment

  • Over 600 already-approved drugs are being screened using this approach

• Targeting the ERO1A pathway:

  • Recent research has identified ERO1A inhibition as a therapeutic strategy

  • SELENON antibodies are crucial for validating this approach in patient-derived samples

  • The drug TUDCA has shown promise in inhibiting ERO1A activity, with functional improvements in mouse and cell models

• Selenomab-drug conjugate development:

  • Leveraging selenocysteine incorporation technology

  • These conjugates demonstrate excellent stability, potency, and selectivity

  • While not yet specifically applied to SELENON-RM, this platform shows potential for targeted delivery of therapeutic compounds

• Future research directions and technical challenges:

  • Improving selenocysteine incorporation efficiency (currently at 2-4 mg/L)

  • Optimizing antibody specificity for particular SELENON isoforms

  • Developing conditional approaches that modulate SELENON function only under specific cellular conditions

How do SELENON antibodies contribute to understanding post-transcriptional regulation of SELENON expression?

SELENON antibodies are instrumental in elucidating the complex post-transcriptional regulatory mechanisms governing SELENON expression:

• Investigation of selenocysteine incorporation mechanisms:

  • SELENON contains selenocysteine (Sec), the 21st amino acid, encoded by the UGA stop codon

  • Antibodies targeting specific regions help evaluate the efficiency of Sec incorporation

  • This has revealed the role of the Sec incorporation sequence (SECIS) and stop-codon redefinition element (SRE) in SELENON translation

• Analysis of RNA editing effects:

  • A-to-I RNA editing mediated by ADAR1 affects SELENON expression by antagonizing Alu exonization

  • Antibodies allow researchers to correlate edited mRNA levels with protein expression

  • This has shown that ADAR1 knockdown increases the inclusion isoform of SELENON mRNA

• Alternative splicing detection:

  • SELENON undergoes alternative splicing, producing multiple isoforms

  • Antibodies targeting different regions help identify specific isoforms

  • Studies show skeletal muscle has the highest frequency of the SELENON skipping isoform

• Methodology for investigating RNA-protein interactions:

  • RNA immunoprecipitation using SELENON antibodies

  • Can reveal interactions with regulatory RNA binding proteins

  • Helps map regulatory elements affecting SELENON expression

• Tissue-specific regulation:

  • Expression analysis using SELENON antibodies revealed tissue-specific regulation

  • Levels of ADAR1 and hnRNP C are lower in skeletal muscle than in other tissues

  • This explains the high accumulation levels of the Alu exon of SELENON mRNA in mature skeletal muscle

What are the current protocols for developing and characterizing neutralizing antibodies against SELENON?

Development of neutralizing antibodies against SELENON involves several methodological steps, drawing from approaches used for related selenoproteins:

• Initial antibody generation strategies:

  • Immunization with purified recombinant SELENON protein or specific peptides

  • Selection of candidate antibodies through binding assays

  • Primary screening for cell surface binding inhibition (as demonstrated with SeP antibodies)

• Functional neutralization screening:

  • Assess antibody effects on SELENON-mediated redox functions

  • Measure impact on calcium dynamics in cell models

  • Evaluate ability to block specific protein-protein interactions

• Epitope mapping methodology:

  • Identify binding regions through techniques such as:

    • Peptide arrays

    • Hydrogen-deuterium exchange mass spectrometry

    • Alanine scanning mutagenesis

  • Focus on functional domains like the first histidine-rich region (FHR)

• Neutralizing antibody characterization:

• Validation in disease models:

  • Test antibody effects in SELENON-deficient cell models

  • Evaluate impact on oxidative stress markers

  • Assess improvement in calcium handling abnormalities

  • Measure functional outcomes in animal models of SELENON-RM

The methodological approach above is based on successful development of neutralizing antibodies against selenoprotein P, which improved glucose intolerance and insulin secretion in a mouse model of diabetes .

What are the optimal protocols for using SELENON antibodies in Western blot applications?

Based on published research using SELENON antibodies, the following protocol optimizations are recommended for Western blot applications:

• Sample preparation:

  • Use fresh samples when possible to minimize protein degradation

  • For tissue samples: homogenize in RIPA buffer containing protease inhibitors, reducing agents, and phosphatase inhibitors

  • For cell lysates: consider using NP-40 or Triton X-100 based lysis buffers with 1-2% detergent concentration

  • Include reducing agents (e.g., DTT or β-mercaptoethanol) in sample buffer to maintain selenocysteine residues

• Gel electrophoresis conditions:

  • Use 8-10% polyacrylamide gels for optimal separation of the ~65.8 kDa SELENON protein

  • Load 20-50 μg of total protein per lane

  • Include positive control samples (skeletal muscle lysate) and molecular weight markers

• Transfer parameters:

  • Semi-dry or wet transfer systems are both suitable

  • Transfer at 100V for 60-90 minutes (wet) or 15-25V for 30-45 minutes (semi-dry)

  • Use PVDF membranes for better protein retention and signal-to-noise ratio

• Blocking and antibody conditions:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Incubate overnight at 4°C for optimal binding

  • Secondary antibody dilutions typically 1:5000 to 1:10000

  • Extended washing steps (5 × 5 minutes) improve signal-to-noise ratio

• Detection considerations:

  • Enhanced chemiluminescence (ECL) provides sufficient sensitivity for most applications

  • Fluorescent secondary antibodies allow for multiplex detection and broader dynamic range

  • When analyzing post-translational modifications, consider parallel blots with phospho-specific or glycosylation-specific detection methods

• Troubleshooting common issues:

How can I optimize immunohistochemistry/immunofluorescence protocols for SELENON detection?

Optimizing protocols for SELENON detection in tissue and cell samples requires attention to several key parameters:

• Fixation optimization:

  • PFA fixation (4%) has been successfully used for SELENON detection

  • Limit fixation time to 10-15 minutes for cells and 24-48 hours for tissues

  • Consider testing acetone fixation (10 minutes at -20°C) as an alternative for certain applications

  • For tissue sections, 4-8 μm thickness provides optimal results

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow slow cooling to room temperature to prevent tissue damage

  • For formalin-fixed tissues, consider additional retrieval with 0.1% SDS in PBS for 5 minutes

• Permeabilization considerations:

  • As an ER-resident protein, SELENON requires effective membrane permeabilization

  • Triton X-100 (0.1-0.3%) has been successfully used in ICC/IF protocols

  • Digitonin (50-100 μg/ml) provides gentler permeabilization that better preserves ER structure

  • Permeabilization time should be optimized (typically 5-15 minutes)

• Blocking and antibody conditions:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to blocking solution for improved penetration

  • For SELENON antibodies, typical dilutions range from 1:50 to 1:500

  • Extended primary antibody incubation (overnight at 4°C) improves specific signal

• Co-staining considerations:

  • Pair SELENON antibodies with ER markers (calnexin, PDI) to confirm localization

  • For oxidative stress studies, combine with antibodies against antioxidant enzymes

  • Use sequentially applied secondary antibodies to minimize cross-reactivity

  • Include DAPI nuclear counterstain for orientation

• Signal detection optimization:

  • Adjust exposure settings to prevent signal saturation

  • Use spectral unmixing for accurate separation of fluorophores

  • Consider signal amplification (tyramide signal amplification) for low abundance detection

  • Z-stack acquisition improves visualization of ER distribution patterns

• Controls and validation:

  • Include tissue samples known to express SELENON (skeletal muscle)

  • Use SELENON-deficient tissues/cells as negative controls

  • Include secondary-only controls to assess background fluorescence

  • Validate results with multiple antibodies targeting different SELENON epitopes

What approaches can be used to detect specific isoforms of SELENON with antibodies?

Detection of specific SELENON isoforms requires careful antibody selection and experimental design:

• Understanding SELENON isoform diversity:

  • SELENON has multiple isoforms resulting from alternative splicing

  • Isoform 1 and isoform 2 are expressed in skeletal muscle, brain, lung, and placenta

  • Isoform 2 shows additional expression in heart, diaphragm, and stomach

  • Alternative splicing related to Alu exonization generates additional variants

• Epitope-specific antibody selection:

  • Choose antibodies targeting regions that differ between isoforms

  • For detecting all isoforms, select antibodies against conserved regions

  • Consider developing custom antibodies against isoform-specific junction peptides

  • Validate antibody specificity using recombinant isoform proteins

• Western blot approaches:

  • Use high-resolution gel systems (gradient gels) to separate similarly sized isoforms

  • Include positive controls of tissues known to express specific isoforms

  • Consider 2D gel electrophoresis to separate isoforms with similar molecular weights but different isoelectric points

  • Use careful sample preparation to maintain post-translational modifications that may be isoform-specific

• RT-PCR validation:

  • Complement antibody-based detection with PCR confirmation of isoform expression

  • Design primers spanning exon junctions specific to each isoform

  • Correlate mRNA and protein expression levels to confirm antibody specificity

  • This approach has been used to detect the inclusion isoform with the Alu exon versus the skipping isoform

• Technical considerations for isoform detection:

• Advanced approaches:

  • Immunoprecipitation followed by mass spectrometry for isoform identification

  • Proximity ligation assays to detect isoform-specific protein interactions

  • Super-resolution microscopy to visualize potential isoform-specific localization patterns

Research has shown that skeletal muscle has the highest frequency of the SELENON skipping isoform, while alternative splicing related to Alu exonization is regulated by ADAR1-mediated RNA editing , making these considerations particularly important for muscle-focused studies.

How might advances in antibody engineering improve SELENON research and therapeutic applications?

Emerging antibody engineering technologies offer significant potential for advancing SELENON research and therapies:

• Site-specific conjugation technologies:

  • Selenomab technology incorporates selenocysteine residues for site-specific conjugation

  • This allows precise control of drug-to-antibody ratios (DAR) and conjugation sites

  • SELENON-targeting antibodies could benefit from this approach for creating stable, homogeneous antibody-drug conjugates

• Bispecific antibody applications:

  • Antibodies that simultaneously target SELENON and other proteins

  • Could facilitate studies of protein-protein interactions

  • Potential therapeutic applications targeting both SELENON and stress response pathways

  • May enable targeted delivery of therapeutic agents to specific cellular compartments

• Intracellular antibody delivery systems:

  • As an ER-resident protein, SELENON requires intracellular targeting

  • Cell-penetrating peptides or lipid nanoparticle delivery systems

  • Exosome-based delivery of SELENON-targeting antibodies

  • These approaches could enable modulation of SELENON function in living cells

• Improvements in humanized antibodies:

  • Reduction of immunogenicity for therapeutic applications

  • Structure-guided humanization preserving critical binding residues

  • These advancements are particularly relevant for potential long-term treatment of chronic SELENON-RM

• Nanobodies and single-domain antibodies:

  • Smaller size enables better tissue penetration and potentially intracellular delivery

  • Higher stability and potentially better access to cryptic epitopes

  • Could access ER-resident SELENON more effectively than conventional antibodies

  • May enable new imaging approaches for tracking SELENON dynamics in living systems

• Computational antibody design:

  • Structure-based design of antibodies with improved specificity

  • Machine learning approaches to predict optimal antibody-epitope interactions

  • These methods could accelerate development of highly specific SELENON-targeting antibodies

The most promising near-term application is likely the development of stable selenomab-drug conjugates for SELENON-RM, as this technology has already demonstrated "excellent stability, potency, and selectivity in diverse in vitro and in vivo models" .

What research gaps remain in our understanding of SELENON function and antibody-based detection methods?

Despite significant progress, several critical knowledge gaps remain in SELENON research that could be addressed with improved antibody tools:

• Structural biology limitations:

  • The complete three-dimensional structure of SELENON remains unresolved

  • Antibodies recognizing specific conformational states could help elucidate functional domains

  • Antibody-assisted cryo-EM studies might reveal structure-function relationships

• Dynamic protein interactions:

  • The complete interactome of SELENON under physiological and stress conditions is not fully mapped

  • Proximity labeling approaches using SELENON antibodies could identify transient interaction partners

  • Time-resolved studies of SELENON interactions during calcium flux remain technically challenging

• Tissue-specific functions:

  • While predominantly studied in muscle, SELENON's role in other expressing tissues is poorly understood

  • Tissue-specific antibody panels would help characterize expression patterns across development

  • The relationship between isoform expression and tissue-specific function requires further investigation

• Pathophysiological mechanisms:

  • How different SELENON mutations affect protein function at the molecular level

  • The relationship between SELENON dysfunction and disease progression

  • The compensatory mechanisms in different tissues affected by SELENON mutations

• Technical limitations:

  • Lack of standardized protocols for quantitative assessment of SELENON levels

  • Limited availability of well-validated antibodies against specific SELENON domains

  • Challenges in distinguishing post-translationally modified forms

• Research needs and future directions:

Recent advances in understanding ERO1A as a counterbalancing partner for SELENON offer promising new directions for therapeutic development , but many fundamental questions about SELENON biology remain unanswered and require improved antibody tools for investigation.