At5g18800 Antibody
Description
Introduction to At5g18800 Antibody
The At5g18800 antibody targets the PGIV protein (AT5G18800), a subunit of mitochondrial Complex I (NADH dehydrogenase) in Arabidopsis thaliana. Complex I is the largest enzyme complex in the oxidative phosphorylation system, responsible for catalyzing NADH-quinone oxidoreduction. This antibody enables the study of Complex I's structure, assembly, and functional roles in plant mitochondria .
Immunogen and Epitope Specificity
The antibody was generated using a synthetic peptide immunogen derived from the C-terminal region of PGIV (Table 1).
Table 1: Immunogen and antigenic properties
Product Characteristics
Form: Lyophilized powder
Storage: -20°C (manual defrost freezer recommended to avoid repeated freeze-thaw cycles)
Recommended applications:
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Western blot (detection limit: 5–25 ng of tagged fusion protein)
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Immunoprecipitation (validated with N- and C-terminal DYKDDDDK-tagged proteins)
Research Applications in Plant Mitochondrial Studies
The antibody has been critical in:
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Complex I assembly analysis: Identification of stable subcomplexes in Arabidopsis mutants using Blue-Native PAGE .
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Subcellular localization: Confirming mitochondrial membrane localization via fractionation studies .
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Comparative genomics: Assessing PGIV conservation across 15 plant species, revealing its essential role in electron transport .
The antibody recognizes PGIV orthologs in:
Comparative Analysis with Other Complex I Subunits
Table 3: Complex I subunit antibody benchmarks
| Subunit | MW (kDa) | Antibody Type | Detection Method | Reference |
|---|---|---|---|---|
| PGIV | 25 | Polyclonal | Western blot, IP | |
| MWFE | 8 | Polyclonal | Chemiluminescence | |
| Nad9 | 23 | Polyclonal | BN-PAGE/NBT staining |
Key Research Findings
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Assembly intermediates: At5g18800 antibody helped identify a 400-kDa subcomplex in Arabidopsis mutants, suggesting PGIV's role in mid-stage Complex I assembly .
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Conservation patterns: The epitope shows 93% sequence conservation between AT3G06310 and AT5G18800 PGIV isoforms, explaining cross-reactivity .
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Functional redundancy: Double mutants of PGIV paralogs (AT3G06310/AT5G18800) show severe growth defects, unlike single mutants .
Technical Considerations
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Sensitivity optimization: 0.2 µg/ml primary antibody concentration achieved clear signals in Western blots with 10 ng antigen .
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Buffer compatibility: Validated in ACA buffer (750 mM aminocaproic acid, 50 mM Tris-HCl pH 7.0) for native complex analyses .
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Artifact prevention: Acetone precipitation at -80°C recommended prior to mass spectrometry downstream processing .
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
FAQs for Researchers on At5g18800 Antibody in Academic Research
The At5g18800 antibody targets the Arabidopsis thaliana NADH-ubiquinone oxidoreductase subunit, a critical component of mitochondrial Complex I. Below are research-focused FAQs addressing experimental design, methodological challenges, and data interpretation.
What advanced techniques are suitable for studying At5g18800 protein-protein interactions?
Methodological Answer:
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Co-immunoprecipitation (Co-IP): Use anti-At5g18800 antibodies with mitochondrial lysates under native conditions. Identify co-precipitated proteins via mass spectrometry .
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Blue Native PAGE (BN-PAGE): Resolve intact Complex I (~980 kDa) and subcomplexes to study assembly defects in Arabidopsis mutants .
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Bimolecular fluorescence complementation (BiFC): Tag At5g18800 and candidate interactors (e.g., At5g41150) to visualize interactions in planta .
Example Workflow:
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Isolate mitochondria using sucrose density gradients.
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Perform BN-PAGE followed by immunoblotting with At5g18800 antibody .
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Compare band patterns between wild-type and at5g18800 mutants to identify disrupted subcomplexes.
How should researchers design experiments to resolve contradictions in At5g18800 functional data?
Methodological Answer:
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Dosage-dependent phenotypic analysis: Compare at5g18800 knockdown (RNAi) vs. knockout lines to distinguish partial vs. complete loss-of-function effects .
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Complementation assays: Express tagged At5g18800 variants (e.g., HA-FLAG) in mutant backgrounds to verify functional rescue .
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Context-specific profiling: Measure NADH dehydrogenase activity under stress (e.g., high light) to assess environmental dependency .
Data Contradiction Case:
If studies report conflicting subcellular localization, repeat immunogold labeling with high-pressure freezing samples to preserve membrane integrity .
What are common pitfalls in quantifying At5g18800 expression via Western blot?
Methodological Answer:
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Mitochondrial loading controls: Normalize to voltage-dependent anion-selective channel (VDAC) instead of cytosolic actin .
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Denaturation optimization: Use 4% SDS in Laemmli buffer to fully solubilize hydrophobic Complex I subunits .
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Signal saturation avoidance: Perform serial dilutions of mitochondrial extracts (e.g., 1:5 to 1:20) to ensure linear detection range .
Troubleshooting Table:
| Issue | Solution | Source |
|---|---|---|
| Weak/no signal | Pre-clear lysates with protein A/G beads | |
| Multiple bands | Include 1% β-mercaptoethanol in sample buffer | |
| High background | Block with 5% BSA + 0.1% Tween-20 |
How can researchers investigate At5g18800’s role in alternative splicing?
Methodological Answer:
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RNA immunoprecipitation (RIP): Crosslink RNA-protein complexes in vivo, immunoprecipitate with At5g18800 antibodies, and sequence bound RNAs .
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Splice variant analysis: Use RT-PCR with primers flanking alternative exons (e.g., At3g12570) in wild-type vs. mutants .
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Co-localization studies: Combine fluorescence in situ hybridization (FISH) for nascent transcripts with At5g18800 immunostaining .
Critical Controls:
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Include IgG isotype controls in RIP experiments.
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Validate splicing defects using independent mutants (e.g., CRISPR-Cas9 lines) .
What strategies improve At5g18800 antibody stability in long-term assays?
Methodological Answer:
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Storage optimization: Aliquot antibodies in PBS + 50% glycerol, store at -80°C. Avoid freeze-thaw cycles .
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Carrier protein addition: Include 1 mg/mL BSA in antibody dilution buffer to prevent aggregation .
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Activity monitoring: Perform monthly endpoint titers using standardized mitochondrial extracts .
