At1g18910 Antibody
Description
Introduction to At1g18910 Antibody
The At1g18910 antibody targets the protein product of the At1g18910 gene, which encodes BTSL2, a plant-specific E3 ubiquitin ligase. BTSL2 functions as a negative regulator of iron uptake by modulating transcription factors involved in Fe deficiency responses .
BTSL2 Protein Structure
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Domains: Contains a RING domain, zinc ribbon, and CHY domain critical for protein interactions .
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Interaction Sites: Residues 450–550 in the C-terminal region mediate binding to transcription factors (e.g., FIT, bHLH subgroup IVc) and the small effector protein FEP3/IMA1 .
Gene Expression
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Induced under Fe-deficient conditions, primarily in root epidermis and cortex cells .
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Coexpressed with Fe uptake genes (e.g., IRT1, FIT) but not with BTS, its homolog .
Genetic Studies
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Mutant Phenotypes:
Mechanistic Role
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BTSL2 ubiquitinates and degrades FIT, a central transcription factor in Fe acquisition .
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Interacts directly with FIT but not bHLH39, suggesting specificity in Fe signaling .
Key Techniques
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Coimmunoprecipitation: Validates BTSL2 interactions with FIT and FEP3/IMA1 .
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Far-Western Blotting: Confirms binding to FIT but not bHLH39 .
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Yeast Two-Hybrid (Y2H) Assays: Maps interaction domains (e.g., C-terminal residues 450–550) .
Table 1: Metal Concentrations in Mutants
| Genotype | Root Fe (μg/g) | Shoot Fe (μg/g) | Root Zn (μg/g) |
|---|---|---|---|
| Wild type | 120 ± 15 | 80 ± 10 | 25 ± 3 |
| btsl1 btsl2 | 210 ± 20* | 150 ± 18* | 45 ± 5* |
| bts-1 btsl1 btsl2 | 290 ± 25* | 200 ± 22* | 60 ± 7* |
| *Data from ; p < 0.05 vs. wild type. |
Table 2: BTSL2 Interaction Assays
Implications and Future Directions
The At1g18910 antibody has been pivotal in elucidating BTSL2’s role in Fe homeostasis. Future work could explore:
Product Specs
Target Background
Q&A
Basic Research Questions
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How do I validate the specificity of At1g18910 antibody in Arabidopsis thaliana studies?
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Method: Use CRISPR-generated knockout (KO) lines (e.g., btsl2 mutants) for immunoblotting comparisons. Validate via:
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Western blotting: Compare band presence/absence in wild-type vs. KO lysates .
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Immunoprecipitation (IP): Test antibody enrichment efficiency using mass spectrometry to confirm target binding .
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Cross-reactivity screening: Include lysates from paralogs (e.g., BTSL1 mutants) to rule out off-target binding .
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What are the primary applications of At1g18910 antibody in plant biology research?
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Key uses:
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Protein localization: Immunofluorescence (IF) in root tissues to study iron regulation .
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Functional studies: Detect BTSL2 protein levels under iron-deficient conditions using quantitative Western blotting .
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Interaction assays: Co-IP with FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) to investigate ubiquitination pathways .
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How do I troubleshoot inconsistent antibody performance across experiments?
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Steps:
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Buffer optimization: Test lysis buffers with varying detergent concentrations (e.g., 0.1–1% Triton X-100) to improve epitope accessibility .
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Post-translational modifications: Use phosphatase/protease inhibitors during protein extraction to preserve target integrity .
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Batch validation: Compare new antibody lots against archived KO/WT lysates to ensure consistency .
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Advanced Research Questions
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How to design experiments assessing BTSL2 functional redundancy with BTSL1 in iron homeostasis?
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Approach:
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Genetic crosses: Generate btsl1 btsl2 double mutants and quantify iron uptake genes (IRT1, FRO2) via qRT-PCR .
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Phenotypic rescue: Express BTSL2 under tissue-specific promoters in double mutants to map spatial roles .
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Single-cell RNA-seq: Profile root epidermal cells to identify compensatory pathways in paralog mutants .
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What methods resolve contradictions in BTSL2 interaction data (e.g., FIT binding vs. degradation)?
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Strategies:
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Pulse-chase assays: Measure FIT protein half-life in btsl2 mutants vs. wild-type .
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In vitro ubiquitination**: Purify BTSL2 and FIT to test E3 ligase activity using ubiquitin-conjugation assays .
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Bimolecular fluorescence complementation (BiFC): Visualize BTSL2-FIT interactions in planta under iron-deficient conditions .
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How to quantify BTSL2 expression dynamics during iron stress?
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Tools:
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Nanostring nCounter: Profile BTSL2 mRNA and protein (via antibody-based detection) in parallel .
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ELISA optimization: Coat plates with anti-BTSL2 antibody and compare chemiluminescent vs. colorimetric detection limits .
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Live-cell imaging: Fuse BTSL2 with fluorescent tags (e.g., GFP) and track degradation kinetics using confocal microscopy .
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Table 1: At1g18910 Antibody Specifications
| Product Code | Host Species | Target Protein | Applications |
|---|---|---|---|
| CSB-PA117109XA01DOA | Rabbit | BTSL2 (AT1G18910) | WB, IP, IF |
Table 2: BTSL2 Functional Interactions
| Interaction Partner | Assay Type | Outcome | Citation |
|---|---|---|---|
| FIT | Far-Western blot | Direct binding via CHY/RING domain | Hindt et al. 2017 |
| BTSL1 | Genetic analysis | Redundant iron regulation | Rodríguez-Celma et al. 2017 |
Methodological Recommendations
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For KO validation: Use YCharOS-validated protocols comparing parental vs. KO cell lines in mosaic imaging assays .
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Phylogenetic analysis: Apply maximum-likelihood trees to BTSL orthologs (e.g., Medtr7g037040) to infer evolutionary conservation .
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Antibody optimization: Humanize Arabidopsis antibodies using frameworks with high stability (e.g., VH3-23/VK1-39) to improve solubility .
