SELENOW Antibody

What is SELENOW and what are its primary biological functions?

SELENOW (also known as SELW, SEPW1, or SelW) functions as a glutathione (GSH)-dependent antioxidant involved in redox-related processes. It appears to play a significant role in myopathies associated with selenium deficiency . Recent research has identified its importance in tau protein regulation, with evidence suggesting it contributes to tau homeostasis and synaptic maintenance in the context of Alzheimer's disease (AD) . SELENOW contains a thioredoxin-like motif with a critical cysteine residue (Cys37) that forms a disulfide linkage with cysteine-322 (C322) of tau protein, which may explain its regulatory effect on tau protein levels .

What types of SELENOW antibodies are currently available for research applications?

Several types of SELENOW antibodies are available for research:

• Unconjugated polyclonal antibodies (such as ABIN7168779) targeting specific amino acid sequences (e.g., AA 11-86)

• Conjugated antibodies with reporter molecules:

  • HRP-conjugated antibodies for enhanced chemiluminescence detection

  • Biotin-conjugated antibodies for amplification systems

  • FITC-conjugated antibodies for fluorescence microscopy

Most commercially available antibodies are raised against recombinant human SELENOW protein and purified using Protein G chromatography to achieve >95% purity .

What are the recommended applications for SELENOW antibodies?

SELENOW antibodies are primarily used in:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of SELENOW in various sample types

• Immunohistochemistry (IHC): For visualization of SELENOW distribution in tissue sections, with recommended dilutions typically ranging from 1:20 to 1:200

• Western blotting: For detection of SELENOW protein levels in tissue or cell lysates

• Co-immunoprecipitation (Co-IP): For studying protein-protein interactions, particularly between SELENOW and tau

• Proximity Ligation Assay (PLA): For detecting and visualizing protein interactions in situ

Each application requires specific optimization depending on the experimental design and sample type.

How should SELENOW antibodies be stored and handled to maintain optimal reactivity?

For maximum antibody stability and performance:

• Store antibodies at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficiency

• For antibodies in liquid format (typically in 50% glycerol, 0.01M PBS, pH 7.4 with 0.03% ProClin 300 as preservative), maintain proper buffer conditions during handling

• Exercise appropriate caution when handling antibody preparations containing ProClin, as this is classified as a hazardous substance that should only be handled by trained personnel

• For long-term storage, consider aliquoting the antibody to minimize freeze-thaw cycles

How can immunohistochemistry protocols be optimized for SELENOW detection in brain tissue?

Optimizing SELENOW detection in brain tissue requires several considerations:

• Fixation method: Paraformaldehyde (4%) fixation generally preserves SELENOW epitopes while maintaining tissue architecture.

• Antigen retrieval: SELENOW epitopes may be masked during fixation. Consider:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Enzymatic retrieval with proteinase K for certain tissue preparations

• Antibody dilution optimization: Begin with the manufacturer's recommended range (1:20-1:200) and perform a dilution series to determine optimal signal-to-noise ratio .

• Detection systems: For low abundance SELENOW, particularly in AD tissue where levels are decreased, amplification systems such as tyramide signal amplification may enhance detection sensitivity .

• Anatomical considerations: SELENOW exhibits region-specific expression patterns. In wild-type mice, it is enriched in axonal processes of hippocampal CA1 neurons, which should be considered when designing experiments and interpreting results .

What considerations are important when using SELENOW antibodies in Alzheimer's disease models?

When studying SELENOW in AD models, researchers should consider:

• Model selection: Different AD models show variable SELENOW expression patterns. Triple transgenic (3×Tg) AD mice exhibit reduced SELENOW in hippocampi compared to wild-type mice .

• Age-dependent changes: SELENOW levels may fluctuate with disease progression. Studies in 8-month-old 3×Tg AD mice show significantly reduced SELENOW compared to age-matched controls .

• Relationship with tau pathology: Since SELENOW levels inversely correlate with tau, antibody-based experiments should include co-staining for phosphorylated tau epitopes (particularly Ser396 and Ser404) .

• Subcellular localization: SELENOW localization changes in pathological conditions. In healthy tissues, it is enriched in axonal processes, but this pattern is disrupted in AD .

• Experimental controls: Include both positive controls (regions with known high SELENOW expression) and negative controls (SELENOW knockout tissues if available) to validate antibody specificity .

How can I verify SELENOW antibody specificity in my experimental system?

Verifying antibody specificity is crucial for reliable results:

• Knockout validation: Ideally, use tissue from SELENOW knockout animals as a negative control to confirm absence of signal .

• siRNA knockdown: In cell culture systems, perform SELENOW siRNA knockdown experiments to confirm signal reduction correlates with protein level reduction .

• Peptide competition: Pre-incubate the antibody with excess recombinant SELENOW protein or immunizing peptide to block specific binding sites.

• Multiple antibody validation: Use antibodies from different sources or those recognizing different epitopes to confirm consistency in detection patterns.

• Cross-reactivity assessment: Test reactivity against closely related selenoproteins (e.g., SELENOV) to ensure specificity, as demonstrated in studies showing SELENOV does not regulate tau in the same manner as SELENOW .

What factors might cause variable SELENOW detection in brain tissue samples?

Variable SELENOW detection may result from:

• Region-specific expression: SELENOW shows differential expression across brain regions, with enrichment in hippocampal CA1 neurons' axonal processes in wild-type mice .

• Disease-related changes: Significant downregulation occurs in the hippocampus and temporal cortex of AD patients and mouse models .

• Selenium status: As a selenoprotein, SELENOW expression is influenced by selenium availability. Variations in dietary selenium may affect experimental results, particularly in longitudinal studies.

• Technical factors:

  • Fixation duration affecting epitope accessibility

  • Ineffective antigen retrieval

  • Suboptimal antibody concentration

  • Inconsistent tissue processing

• Age and sex differences: Consider age-matched controls and account for potential sex-based differences in SELENOW expression.

How can I address potential cross-reactivity with other selenoproteins?

To minimize cross-reactivity issues:

• Epitope selection: Choose antibodies targeting unique regions of SELENOW that have minimal sequence homology with other selenoproteins.

• Validation experiments: Perform side-by-side comparisons with other selenoproteins, particularly those with TXN-like motifs such as SELENOV, which has been experimentally shown not to affect tau levels despite structural similarities to SELENOW .

• Immunoprecipitation followed by mass spectrometry: Identify all proteins pulled down by your antibody to assess potential cross-reactivity.

• Blocking peptide experiments: Use synthetic peptides corresponding to the immunogen sequence to block specific binding and identify non-specific signals.

• Western blot analysis: Confirm that your antibody detects a single band of the expected molecular weight (~10 kDa for SELENOW).

How can SELENOW antibodies be used to study tau-SELENOW interactions?

SELENOW antibodies can be employed in several techniques to investigate tau-SELENOW interactions:

• Co-immunoprecipitation (Co-IP): Use SELENOW antibodies to pull down protein complexes and probe for tau, or vice versa. This approach has confirmed binding between SELENOW and tau in previous studies .

• Proximity Ligation Assay (PLA): This technique can visualize protein-protein interactions with spatial resolution, identifying where in the cell SELENOW and tau interact.

• Bimolecular Fluorescence Complementation (BiFC): By tagging SELENOW and tau with complementary fluorescent protein fragments, their interaction can be visualized when the fragments come together.

• FRET/FLIM analysis: Use fluorescently labeled antibodies to detect energy transfer between SELENOW and tau, indicating close proximity.

• Competitive binding assays: Study how SELENOW competes with Hsp70 for tau binding, which has been identified as a potential mechanism for SELENOW's effect on tau degradation .

What approaches can be used to investigate SELENOW's role in UPS-mediated tau degradation?

To study SELENOW's involvement in ubiquitin-proteasome system (UPS) tau degradation:

• Proteasome inhibition experiments: Use inhibitors like MG132 to block proteasomal degradation and observe effects on tau levels in the presence/absence of SELENOW .

• Protein half-life studies: Perform cycloheximide chase experiments to measure tau protein stability with varying SELENOW levels.

• Ubiquitination assays: Use antibodies against ubiquitin and tau to assess whether SELENOW affects tau ubiquitination status.

• Degradation pathway inhibitor panels: Apply selective inhibitors of different degradation pathways (proteasomal, lysosomal, autophagy) to determine which mechanism is primarily affected by SELENOW.

• Fluorescent reporters: Utilize fluorescent UPS reporter systems in combination with SELENOW manipulation to visualize real-time effects on proteasomal activity.

How can I evaluate the effect of SELENOW on tau post-translational modifications using antibodies?

To assess SELENOW's impact on tau post-translational modifications (PTMs):

• Phospho-specific tau antibodies: Use antibodies targeting specific phosphorylation sites, particularly Ser396 and Ser404, which show significant changes in response to SELENOW overexpression .

• Mass spectrometry: Combine immunoprecipitation with mass spectrometry to comprehensively map tau PTMs affected by SELENOW.

• Phosphatase inhibition experiments: Use phosphatase inhibitors to determine if SELENOW affects tau phosphorylation or dephosphorylation pathways.

• Tau isoform-specific analyses: Examine SELENOW's differential effects on 3-repeat versus 4-repeat tau isoforms using isoform-specific antibodies .

• Kinase activity assays: Investigate whether SELENOW modulates the activity of tau kinases (CDK5, GSK3β, etc.) using activity-specific antibodies.

How should changes in SELENOW expression be interpreted in the context of Alzheimer's disease models?

When interpreting SELENOW expression changes in AD models:

• Inverse correlation with tau: Decreased SELENOW levels typically correlate with increased tau aggregation and phosphorylation. This relationship appears to be causal, as SELENOW manipulation directly affects tau levels .

• Regional specificity: Assess changes in context of brain region. SELENOW reduction in hippocampus and temporal cortex of AD patients and mouse models is particularly significant .

• Functional implications: Connect SELENOW changes to functional outcomes, as SELENOW overexpression in 3×Tg AD mice ameliorates learning and memory deficits without affecting locomotion or anxiety .

• Pathological correlates: Relate SELENOW levels to specific AD pathological features:

  • Neurofibrillary tangle density (reduced with SELENOW overexpression)

  • Tau phosphorylation status (particularly at Ser396 and Ser404)

  • Neuroinflammatory markers (Iba-1 reduction with SELENOW overexpression)

  • Oligodendrocyte markers (Oligo2 upregulation with SELENOW overexpression)

• Temporal dynamics: Consider disease stage when interpreting SELENOW changes, as effects may vary throughout disease progression.

What methodological approaches can help reconcile contradictory findings about SELENOW in different experimental systems?

To address contradictory findings: