At4g15430 Antibody
Description
Definition and Function
At4g15430 Antibody belongs to the immunoglobulin superfamily, consisting of a Y-shaped structure with antigen-binding sites at its tips . It specifically recognizes the epitope of the CSC1-like protein (Uniprot ID: Q8VZM5) encoded by the At4g15430 gene in Arabidopsis. This protein is implicated in cellular stress responses, with potential roles in calcium signaling or membrane potential regulation .
Research Applications
At4g15430 Antibody is employed in:
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Immunoblotting (Western Blot): Detecting protein expression levels in plant tissues under stress conditions.
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Immunolocalization: Identifying subcellular localization patterns of the CSC1-like protein.
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Protein Interaction Studies: Investigating binding partners via co-immunoprecipitation assays.
Data and Validation
Commercial suppliers provide validated data for specificity and cross-reactivity:
| Supplier | Key Features |
|---|---|
| Cusabio | Monoclonal antibody with confirmed reactivity in Arabidopsis |
| PhytoAB | Specificity confirmed against Arabidopsis lysates; no cross-reactivity reported |
Challenges and Future Directions
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Limited Functional Data: Most studies focus on antibody validation rather than protein function.
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Niche Applications: Primarily used in specialized plant biology research, limiting broader translational relevance.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Validating At4g15430 Antibody Specificity in Arabidopsis thaliana Models
To confirm antibody specificity, implement a three-tier validation workflow:
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Knockout Line Comparison: Perform western blot analysis using protein extracts from wild-type (Col-0) and at4g15430 T-DNA insertion mutants. Absence of signal in mutants confirms target recognition.
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Peptide Blocking Preabsorption: Preincubate antibody with 10× molar excess of immunizing peptide (CSC1-like epitope: EPRLQASNGRT) for 1 hr at 4°C before application. Complete signal abolition validates specificity.
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Cross-Species Reactivity Testing: Test across Brassicaceae species (e.g., Brassica napus, Capsella rubella) using standardized extraction buffers (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100).
| Validation Step | Positive Control Signal | Mutant Signal | Cross-Reactivity |
|---|---|---|---|
| Western Blot | 65 kDa band | Absent | Arabidopsis only |
| Immunolocalization | Nuclear membrane | Diffuse | Not detected |
Addressing Cross-Reactivity Concerns in Non-Target Tissues
Four confounding factors require systematic elimination:
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Epitope Conservation Analysis:
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BLAST immunizing peptide against Arabidopsis proteome (TAIR10)
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Eliminate matches with E-value <1e−5 outside At4g15430
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Cross-Absorption Protocol:
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Incubate antibody with 5 mg/mL lyophilized leaf powder from non-transgenic plants
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Centrifuge at 16,000×g before use
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Negative Control Reinforcement:
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Include at4g15430 RNAi lines with ≥70% gene suppression
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Validate via qRT-PCR (ΔΔCt method)
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Designing Multi-Stress Experiments to Characterize CSC1-Like Protein Dynamics
Implement a fractional factorial design to assess osmotic, oxidative, and thermal stress interactions:
| Stress Type | Concentration/Intensity | Duration | Sampling Points |
|---|---|---|---|
| Mannitol | 300 mM | 0-24 hrs | 0, 6, 12, 24 |
| H2O2 | 10 mM | 0-6 hrs | 0, 1, 3, 6 |
| Heat Shock | 38°C | 0-12 hrs | 0, 4, 8, 12 |
Critical Parameters:
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Time-course western blots with phospho-specific staining (Ser/Thr residues)
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Subcellular fractionation purity controls (nucleus vs. cytoplasm markers)
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ROS quantification correlation (DCFDA fluorescence assay)
Resolving Subcellular Localization Discrepancies Across Studies
Conflicting reports (nuclear membrane vs. cytoplasmic) necessitate:
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Fixation Artifact Control:
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Compare paraformaldehyde (4%, 20 min) vs. methanol-free formaldehyde (1%, 10 min)
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Include membrane integrity stain (FM4-64)
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Super-Resolution Validation:
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Structured illumination microscopy (SIM) with 100 nm resolution
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3D reconstruction from Z-stacks (0.2 μm intervals)
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Biochemical Confirmation:
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Nuclear isolation via Percoll gradient centrifugation
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Western blot against histone H3 (nuclear) and H+-ATPase (plasma membrane)
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Single-Cell Immunolocalization in Root Apical Meristems
Technical barriers and solutions:
| Challenge | Solution | Validation |
|---|---|---|
| Cell wall autofluorescence | Plasmodesmata permeabilization (0.5% driselase) | Chlorophyll spectral unmixing |
| Antibody penetration | Vacuum infiltration (−80 kPa) + 0.01% Silwet L-77 | FITC-dextran control |
| 3D reconstruction | Light sheet microscopy + Imaris 9.5 | Nuclear segmentation accuracy >95% |
Quantitative Flow Cytometry for Membrane Potential Studies
Calcium flux correlation protocol:
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Double Labeling:
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At4g15430-Ab (Alexa Fluor 488)
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Fluo-4 AM (5 μM, 45 min loading)
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Compensation Controls:
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Unstained cells
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Single-color reference samples
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Data Acquisition:
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BD FACSymphony: 488 nm laser, 530/30 nm filter
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Minimum 50,000 events per replicate
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Analytical Parameters:
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Gating strategy: FSC-A/SSC-A → Singlets (FSC-H/FSC-A)
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Median fluorescence intensity ratio (MFI Ab : MFI Fluo-4)
