At5g45950 Antibody
Description
Research Context: Functional Insights of At5g45950
The At5g45950 protein is linked to enzymatic activities with potential roles in plant defense. Research highlights include:
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Enzyme Group: Classified as a GDSL-motif esterase/acyltransferase/lipase, suggesting involvement in lipid metabolism or pathogen-related processes .
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Systemic Immunity: Functional studies in Arabidopsis indicate that related proteins (e.g., AZI1) are involved in systemic acquired resistance (SAR), mediated by signaling molecules like azelaic acid . While At5g45950’s direct role in immunity remains under investigation, its enzymatic profile aligns with defense-related pathways.
Hypothetical Applications of the Antibody
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Protein Localization: Tracking At5g45950 in subcellular compartments (e.g., plastids, membranes) during stress responses.
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Protein-Interaction Studies: Identifying binding partners via immunoprecipitation.
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Expression Profiling: Quantifying protein levels under biotic stress (e.g., pathogen infection).
Research Gaps and Future Directions
Current data on the At5g45950 antibody are sparse, reflecting its niche application in plant biology. Key areas for future research include:
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Mechanistic Studies: Elucidating the enzyme’s substrate specificity and catalytic role in lipid metabolism or defense.
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Antibody Optimization: Developing recombinant or monoclonal variants for higher sensitivity.
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Cross-Species Reactivity: Testing utility in closely related plant species (e.g., Arabidopsis lyrata).
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
Basic Research Questions
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What experimental controls are essential when validating At5g45950 antibody specificity in Arabidopsis studies?
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Use knockout mutants (e.g., T-DNA insertion lines) as negative controls to confirm antibody binding depends on the target protein.
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Include pre-immune serum or pre-adsorption with recombinant protein to rule out nonspecific binding.
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Validate via Western blotting against purified At5g45950 protein or lysates from overexpression lines.
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How does tissue-specific extraction impact At5g45950 detection in immunolocalization assays?
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Fixation methods: Use formaldehyde-based fixatives for root tips (preserves epitopes) vs. cryofixation for mature leaves (avoids phenolic interference).
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Protease inhibitors: Add 1 mM PMSF or 1× cOmplete™ protease inhibitor cocktail to extraction buffers for meristematic tissues.
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Advanced Research Questions
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How to reconcile contradictory subcellular localization data for At5g45950 across studies?
Factor Nuclear Signal Reported Cytoplasmic Signal Reported Growth conditions Low nitrogen High sucrose Antibody dilution 1:500 1:200 Fixation time 20 min 60 min -
Resolution strategy: Standardize protocols using the Arabidopsis Cell Atlas guidelines and perform colocalization with organelle markers (e.g., histone H2B for nuclei).
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What statistical approaches are optimal for quantifying At5g45950 expression gradients in root apical meristems?
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Spatial analysis: Apply MorphoGraphX software to segment cells and calculate fluorescence intensity per cell layer.
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Normalization: Use UBQ10-GFP as a reference for batch-to-batch variability.
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Methodological Challenges
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How to optimize ChIP-seq protocols for At5g45950 in chromatin-bound vs. soluble fractions?
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Crosslinking: Test 1% formaldehyde for 15 min vs. dual crosslinking (formaldehyde + EGS).
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Sonication: 5 cycles of 30 sec pulses (output 4) for chromatin shearing, verified via agarose gel.
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What metrics indicate antibody degradation in long-term Arabidopsis time-course experiments?
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Performance benchmarks:
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≥ 70% signal reduction in knockout controls
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CV < 15% across technical replicates
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Storage: Aliquot in 50% glycerol at -80°C; avoid >3 freeze-thaw cycles.
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Data Interpretation
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How to distinguish technical artifacts from biological variability in At5g45950 western blot bands?
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Artifact signatures:
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Smearing → Overloaded lysates or protease degradation
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Extra bands → Incomplete blocking or secondary antibody cross-reactivity
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Confirmatory assays: Repeat with native PAGE or 2D gels to assess post-translational modifications.
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What orthogonal techniques validate At5g45950 antibody specificity when genetic mutants are unavailable?
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CRISPR-Cas9-edited lines (≥2 independent lines)
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siRNA knockdown in protoplasts + qRT-PCR correlation (R² > 0.85)
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