At3g51890 Antibody
Description
Functional Role of At3g51890 (CLC3) in Arabidopsis
Clathrin complexes in Arabidopsis consist of three heavy chains (CHC1, CHC2) and three light chains (CLC1, CLC2, CLC3). At3g51890 encodes CLC3, which localizes to the trans-Golgi network/early endosomes (TGN/EE) and plasma membrane (PM) . Key findings include:
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Post-Golgi Trafficking: CLC3 interacts with adaptor protein complex 1 (AP-1) to regulate clathrin recruitment at TGN/EE. AP-1 mutants (e.g., ap1μ2) show 50–60% reduction in TGN/EE-associated clathrin levels .
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Endocytosis Modulation: Loss of AP-1 reduces clathrin-mediated endocytosis (CME) of FM4-64 dye by 40–60% in root cells, demonstrating CLC3's role in vesicle internalization .
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Compensatory Mechanisms: Downregulation of CLC3 increases CHC2 and CLC2 expression, suggesting functional redundancy among clathrin isoforms .
Western Blot and Immunolocalization
The antibody detects endogenous CLC3 in Arabidopsis extracts, with validation via:
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Colocalization studies using GFP-tagged CLC3 and CHC2 in ap1μ2 mutants .
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Quantitative immunofluorescence showing reduced TGN/EE clathrin signals in AP-1-deficient plants .
Key Experimental Findings
Characterization and Validation Challenges
While the At3g51890 antibody is widely used, its validation aligns with broader challenges in antibody reliability:
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Specificity: Cross-reactivity with CLC1/CLC2 is mitigated by antigen-affinity purification .
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Reproducibility: Studies emphasize the need for multiple validation methods (e.g., GFP tagging, mutant analysis) to confirm antibody specificity .
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Storage Stability: Glycerol-based formulation ensures long-term stability but requires strict adherence to -20°C/-80°C storage .
Comparative Analysis of Clathrin Isoforms
| Isoform | Gene ID | Localization | Functional Role |
|---|---|---|---|
| CLC1 | At2g20760 | PM, TGN/EE | Constitutive endocytosis |
| CLC2 | At2g40060 | TGN/EE, chloroplasts | Stress-induced vesiculation |
| CLC3 | At3g51890 | TGN/EE, PM | AP-1–dependent trafficking |
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- At3g51890 plays a crucial role in regulating clathrin-mediated trafficking, auxin signaling, and developmental processes in Arabidopsis. PMID: 23424247
Q&A
Here’s a structured collection of research-focused FAQs for At3g51890 antibody, synthesized from peer-reviewed studies and technical methodologies:
What experimental designs mitigate cross-reactivity in low-abundance target detection?
Advanced strategies:
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Signal amplification: Use tyramide-based systems for weak epitopes while maintaining spatial resolution .
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Multiplex validation: Pair antibody staining with fluorescent protein-tagged At3g51890 transgenic lines for co-localization analysis .
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Quantitative thresholds: Establish cutoff values for signal intensity using RNAi knockdown lines (e.g., ≤30% residual expression as baseline) .
How to resolve contradictory subcellular localization data across studies?
Analytical framework:
What orthogonal methods confirm functional relevance of At3g51890 antibody findings?
Integrated validation pipeline:
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Genetic: Compare phenotypes of CRISPR-Cas9 knockout lines with antibody-based protein depletion .
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Biochemical: Co-immunoprecipitation followed by mass spectrometry to identify interaction partners .
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Biophysical: Surface plasmon resonance (SPR) to quantify binding kinetics (KD ≤10 nM recommended for high-affinity interactions) .
How to optimize antibody concentration for different experimental setups?
Titration guidelines:
What statistical approaches address batch-to-batch variability in long-term studies?
Quality control protocol:
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Normalize signals to reference standards (e.g., recombinant At3g51890-His tagged protein) included in each experiment .
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Implement multivariate ANOVA to account for:
How to map conformational epitopes recognized by At3g51890 antibody?
Structural biology pipeline:
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Phage display mutagenesis: Identify critical residues using alanine-scanning libraries .
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HDX-MS: Map solvent-accessible regions via hydrogen-deuterium exchange .
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In silico docking: Predict binding interfaces using RosettaAntibody with experimental constraints .
What controls are essential for abiotic stress response studies?
Experimental design checklist:
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Stress-specific controls:
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NaCl (150 mM) for osmotic stress
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Methyl viologen (10 μM) for oxidative stress
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Temporal controls: Pre-stress vs. 6/12/24 hr post-treatment timepoints
How to validate antibody performance in protein complex studies?
Stepwise verification:
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Size-exclusion chromatography: Compare elution profiles with/without crosslinkers .
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Blue native PAGE: Confirm co-migration with known complex subunits .
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Single-particle tracking: Quantify dwell times on membrane microdomains .
