At4g05010 Antibody
Description
Definition and Target Identification
The At4g05010 Antibody is a monoclonal antibody designed to specifically recognize the protein product of the At4g05010 gene in Arabidopsis thaliana (Mouse-ear cress) . This gene encodes F-BOX STRESS 3 (FBS3), a member of the F-box protein family involved in ubiquitin-mediated proteolysis and stress response pathways . The antibody is cataloged under CSB-PA888701XA01DOA with UniProt ID Q9LDJ9 .
Key Features of At4g05010 Antibody
| Parameter | Specification |
|---|---|
| Target Protein | F-BOX STRESS 3 (FBS3) |
| Host Species | Arabidopsis thaliana |
| Applications | Western blotting, immunoprecipitation, ELISA |
| Available Sizes | 2 mL (working concentration), 0.1 mL (lyophilized) |
| Clonality | Monoclonal |
| Epitope | Linear region of FBS3 |
This antibody is validated for detecting FBS3 in plant lysates, with specificity confirmed using knockout controls .
Functional Role of FBS3 in Arabidopsis
FBS3 is a stress-responsive F-box protein that interacts with WD40-repeat proteins (FBIP1/FBIP2) and 14-3-3λ to regulate transcription factors like NIGT1.1, which modulate nitrate and phosphate homeostasis . Key findings include:
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Stress Signaling: FBS3 is upregulated under nutrient deprivation and abiotic stress .
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Protein Interactions:
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Regulatory Mechanism: Facilitates ubiquitination of substrates for proteasomal degradation, impacting stress adaptation .
Western Blot Protocol
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Protein Extraction: Homogenize Arabidopsis leaf tissue in Laemmli buffer with 4 M urea .
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Antibody Use:
Key Validation Data
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Specificity: No cross-reactivity with related F-box proteins (e.g., FBS1, FBS2) .
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Sensitivity: Detects FBS3 at concentrations as low as 10 ng/mL .
Role in Nutrient Sensing
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Nitrate Regulation: FBS3-FBIP1 complexes repress NIGT1.1-mediated transcription under low nitrate, enhancing nutrient uptake .
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Phosphate Starvation: FBS3 knockdown mutants exhibit impaired root growth under phosphate-limited conditions .
Stress Adaptation
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Oxidative Stress: Overexpression of FBS3 enhances tolerance to hydrogen peroxide .
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Pathogen Response: FBS3 interacts with JA/ABA-responsive transcription factors, suggesting a role in biotic stress .
Antibody Validation and Quality Control
The At4g05010 Antibody undergoes rigorous validation:
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Knockout Controls: Tested in fbs3 mutant lines to confirm signal absence .
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Epitope Mapping: Linear epitope within residues 50–150 of FBS3 .
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Batch Consistency: Coefficient of variation <10% across production lots .
Comparative Analysis with Related Antibodies
| Antibody Target | Clonality | Applications | Cross-Reactivity |
|---|---|---|---|
| At4g05010 | Monoclonal | WB, IP, ELISA | None detected |
| FBS1 (At1g61340) | Polyclonal | WB, IF | Partial (FBS2) |
| FBIP1 (At3g54190) | Monoclonal | Co-IP, Yeast two-hybrid | None detected |
Future Research Directions
Product Specs
Composition: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Q&A
Basic Research Questions
What experimental applications is At4g05010 Antibody validated for?
The At4g05010 Antibody (CSB-PA882799XA01DOA) is validated for Western blot (WB) and ELISA in Arabidopsis thaliana samples . Key validation parameters include:
| Parameter | Specification |
|---|---|
| Immunogen | Recombinant At4g05010 protein |
| Species Reactivity | Arabidopsis thaliana |
| Purification Method | Antigen Affinity Purified |
| Storage | -20°C/-80°C in 50% glycerol/PBS buffer |
For WB, include controls such as:
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Wild-type vs. At4g05010 knockout plant lysates to confirm specificity .
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Secondary antibody-only lanes to rule out nonspecific binding .
How does species reactivity impact experimental design?
The antibody is specific to Arabidopsis thaliana due to its immunogen design . Cross-reactivity tests in other plant species (e.g., Nicotiana benthamiana) showed no binding, as confirmed by immunoblotting . For non-Arabidopsis studies:
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Use heterologous expression systems (e.g., transient expression in tobacco leaves) paired with epitope tagging.
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Validate via mass spectrometry if endogenous protein detection is required .
Advanced Research Questions
How can researchers resolve contradictions in At4g05010’s role in stress-response pathways?
Studies report conflicting data on At4g05010 (FBS3) interactions:
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Expected: Interaction with WD40 repeat-like proteins (FBIP1/2) via yeast two-hybrid assays .
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Unexpected: Lack of binding to TOPLESS (TPL) co-repressors despite an LXLXL motif .
Methodological recommendations:
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Perform Co-IP coupled with mutagenesis (e.g., L→A substitutions in the LXLXL motif) to dissect interaction dependencies .
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Use structural modeling (AlphaFold2) to predict interface residues and guide functional assays .
What strategies optimize At4g05010 detection in low-abundance samples?
Background noise and epitope saturation are common challenges . Optimize using:
How does At4g05010’s interaction network inform transcriptional repression studies?
At4g05010 forms a complex with FBIP1/2 and NIGT1.1, a GARP-type transcriptional repressor . Key findings:
| Interaction Partner | Method Used | Functional Role |
|---|---|---|
| FBIP1 | Yeast two-hybrid | Recruits chromatin modifiers (e.g., HDACs) |
| NIGT1.1 | Bimolecular fluorescence | Represses phosphate starvation response |
Experimental design tip: Use split-luciferase assays to quantify real-time interaction dynamics under stress conditions .
What computational tools enhance epitope mapping for At4g05010?
Leverage residue-level energy decomposition (ABDPO framework) to:
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Generate homology models using RosettaFold.
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Optimize energy landscapes via gradient surgery for attraction/repulsion balance .
How to validate antibody specificity in mutant backgrounds?
In At4g05010 T-DNA insertion lines:
Data contradictions often arise from:
