SD129 Antibody
Description
Overview of SD129 Antibody
The SD129 antibody, specifically clone SD129-5, is a mouse monoclonal antibody targeting the S2 subunit of the Porcine Epidemic Diarrhea Virus (PEDV) spike (S) protein. It is primarily utilized in virology and immunology research to study viral entry mechanisms, surface protein dynamics, and vaccine development strategies for PEDV, a pathogen causing severe enteric disease in pigs .
Target Specificity and Applications
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Target: S2 subunit of PEDV S protein (a type I transmembrane glycoprotein critical for viral entry).
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Applications:
Mechanistic Insights
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SD129-5 was instrumental in identifying how cytoplasmic tail (CT) motifs (YxxΦ and KVHVQ) regulate S protein trafficking and virion assembly:
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Mutants lacking YxxΦ (e.g., Δ10aa, YA) exhibited 2.5–3x higher surface S protein expression compared to wild-type strains .
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These mutants induced larger syncytia (cell-cell fusion structures) due to enhanced proteolytic activation of surface S proteins .
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Deletion of both motifs (Δ10aa mutant) reduced S protein incorporation into virions by >50%, as visualized by TEM .
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Surface Expression and Syncytium Formation
| Mutant | Surface S Protein Level (vs. WT) | Syncytium Size (Nuclei Count) | Virion S Projections (TEM) |
|---|---|---|---|
| Wild-Type (WT1) | Baseline | 15 ± 3 | 12 ± 2 |
| Δ10aa | 2.8x ↑ | 42 ± 7 | 4 ± 1 |
| YA | 2.5x ↑ | 38 ± 5 | 8 ± 2 |
| Δ5aa | 0.6x ↓ | 2 ± 1 | 10 ± 2 |
Data derived from fluorescence imaging and TEM analysis .
Implications for Vaccine Development
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Mutants with enhanced surface S expression (e.g., Δ10aa) show potential as live-attenuated vaccine candidates due to:
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SD129-5-enabled studies confirmed that CT truncations do not affect neutralizing epitopes, preserving immunogenicity .
Technical Performance
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Staining Protocol: Cells fixed with 4% formaldehyde, stained without permeabilization .
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Quantitative Analysis: Surface/total S protein ratios calculated using fluorescence intensity (green/mCherry) .
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Specificity: No cross-reactivity reported with unrelated viral antigens in TEM or immunoassays .
Limitations and Future Directions
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Limited data on SD129-5’s cross-reactivity with other coronaviruses.
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Further studies needed to evaluate its utility in diagnostic assays or therapeutic antibody engineering.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- SD1-29 mediates lipopolysaccharide sensing in Arabidopsis thaliana. PMID: 25729922
Q&A
Here’s a structured FAQ for SD129 Antibody (phosphorylated α-synuclein at Ser129) tailored to academic research scenarios, integrating technical and methodological insights:
What experimental applications is SD129 Antibody validated for?
SD129 Antibody is validated for:
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Western blot: Detects phosphorylated α-synuclein (p-α-syn) at 17 kDa in denatured mouse/human brain lysates .
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Immunohistochemistry: Identifies Lewy bodies (LBs) and neurites (LNs) in PD patient brain sections (e.g., amygdala) .
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Immunofluorescence: Labels cytoplasmic p-α-syn aggregates in preformed fibril (PFF)-treated neurons .
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ELISA: Quantifies p-α-syn levels with high specificity for phosphorylated epitopes (no cross-reactivity with non-phosphorylated α-syn) .
Methodological Note: Validate antibody specificity using α-syn knockout (KO) controls and compare performance with commercial antibodies (e.g., Wako p-α-syn#1, Abcam p-α-syn#2) .
How should tissue samples be prepared to avoid artifacts in SD129 Antibody staining?
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Fixation: Use 4% paraformaldehyde for <24 hours to preserve epitope integrity.
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Antigen retrieval: Optimize with citrate buffer (pH 6.0) and heat-mediated retrieval to expose phosphorylated epitopes .
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Blocking: 5% BSA in PBS + 0.1% Triton X-100 minimizes non-specific binding in mouse/human brain sections .
Critical Step: Avoid SDS-PAGE denaturation for native conformation studies, as denatured proteins may expose non-specific epitopes (e.g., cross-reactivity with pSer473 neurofilament light chain) .
How do experimental results using SD129 Antibody reconcile discrepancies in p-α-syn quantification across studies?
Example: In Western blots, SD129 may cross-react with denatured proteins in α-syn KO mice (Fig. 4G–I ). Pair with KO controls and orthogonal methods (e.g., immunogold EM) to confirm specificity.
What strategies optimize SD129 Antibody use in mechanistic studies of α-syn aggregation?
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Model Systems:
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Co-localization Studies: Combine with mitochondrial markers (e.g., TOM20) to study p-α-syn deposition on outer mitochondrial membranes .
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Longitudinal Design: Track p-α-syn dynamics using time-resolved immunofluorescence in PFF-treated neurons .
Data Interpretation: Normalize p-α-syn signals to total α-syn (e.g., Santa Cruz α-syn#1 antibody) to account for expression variability .
How does SD129 Antibody performance compare to commercial alternatives in detecting pathogenic α-syn strains?
| Parameter | SD129 Antibody | p-α-syn#1 (Wako) | p-α-syn#2 (Abcam) |
|---|---|---|---|
| Specificity (native) | High (no KO cross-reactivity) | High | Moderate (off-target bands in WB) |
| Sensitivity (WB) | 200 ng detection limit | 200 ng | 500 ng |
| Mitochondrial colocalization | Yes (immunogold EM) | Not tested | Not tested |
Recommendation: Use SD129 for native-state assays (IHC/IF) and pair with p-α-syn#1 for denatured WB .
How to address non-specific signals in SD129 Antibody-based assays?
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WB Artifacts: Excise non-specific bands (e.g., 30–60 kDa) for LC-MS/MS identification to rule out cross-reactivity .
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IHC Background: Pre-adsorb the antibody with non-phosphorylated α-syn peptide (10:1 molar ratio) for 1 hour .
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ELISA Optimization: Use 1:5,000 dilution in 1% BSA/PBS-T to reduce background in human CSF samples .
