EXPA1 Antibody
Description
Definition and Biological Context of EXPA1 Antibody
The EXPA1 Antibody is a polyclonal or monoclonal antibody designed to specifically bind to the EXPA1 protein, a member of the α-expansin family in plants (Arabidopsis thaliana). Expansins are cell wall-loosening proteins that mediate pH-dependent modifications to cell wall structure, enabling cell growth and differentiation . EXPA1 is critical for root development, particularly in lateral root initiation and cell wall stiffness regulation .
Key Features of EXPA1
| Property | Description |
|---|---|
| Gene | EXPA1 (AT1G69530 in Arabidopsis) |
| Protein Function | Cell wall loosening via disruption of cellulose-xyloglucan interactions |
| Hormonal Regulation | Auxin and cytokinin-responsive; ARF5 and ARR1 transcription factors involved |
| Tissue Localization | Root primordia, pericycle cells, and transition zone (TZ) cells |
Applications of EXPA1 Antibody in Research
The EXPA1 Antibody serves as a molecular tool for studying expansin-mediated processes in plant biology. Below are its primary applications, supported by experimental findings:
Localization of EXPA1 Protein
The antibody enables spatial mapping of EXPA1 expression via immunohistochemistry or immunofluorescence. For example:
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Root Development: EXPA1 is expressed in pericycle cells during lateral root initiation, where it promotes asymmetric cell divisions .
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Cell Wall Dynamics: EXPA1 overexpression correlates with increased cell wall stiffness, as measured by Brillouin imaging in Arabidopsis roots .
Quantitative Analysis of EXPA1 Expression
Western Blotting and ELISA are used to quantify EXPA1 levels under varying conditions:
| Experimental Condition | EXPA1 Expression | Source |
|---|---|---|
| Auxin treatment (10 µM NAA) | 5–10-fold upregulation | |
| Cytokinin treatment (5 µM BAP) | 3–4-fold upregulation | |
| expa1-1 mutant | ~95% reduction |
Functional Studies in Hormone Signaling
EXPA1 antibodies help elucidate cross-talk between auxin, cytokinin, and cell wall remodeling:
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Auxin-Dependent Effects: EXPA1 regulates apoplastic pH and cell wall stiffness, enabling auxin-induced root growth .
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Cytokinin-Mediated Rescue: EXPA1 expression in the transition zone (TZ) partially compensates for cytokinin-induced cell differentiation defects .
EXPA1 in Lateral Root Formation
Mutant analysis (expa1-1) revealed that EXPA1 is required for coordinated pericycle cell divisions during lateral root initiation. Key observations:
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Phenotype: Disrupted asymmetric divisions and aberrant root primordia formation .
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Rescue Experiment: Overexpression of EXPA1 in expa1-1 restores wild-type root morphology .
Cell Wall Stiffness and Root Growth
EXPA1 modulates mechanical properties of the cell wall:
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Mechanism: Facilitates cellulose-xyloglucan network rearrangement, reducing cell wall rigidity .
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Impact: expa1-1 mutants exhibit stiffer cell walls, leading to impaired root growth under acidic conditions .
Hormonal Regulation of EXPA1
EXPA1 expression is tightly regulated by auxin and cytokinin pathways:
| Hormone | Regulation | Transcription Factor |
|---|---|---|
| Auxin | Direct activation via ARF5 | ARF5 |
| Cytokinin | Indirect regulation via ARR1/ARR10/ARR12 | ARR1, ARR10, ARR12 |
Specificity and Validation
While EXPA1 antibodies are critical, challenges remain:
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Cross-Reactivity: Potential binding to other α-expansins (e.g., EXPA15) .
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Validation: Requires orthogonal methods (e.g., CRISPR-Cas9 knockout) to confirm specificity .
Emerging Technologies
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- AtEXPA1: Regulates stomatal opening by modifying guard cell wall structure. PMID: 21593593
- Evidence suggests a role for a guard cell expansin (AtEXPA1) in regulating stomatal movement. PMID: 20976459
Q&A
Here’s a structured collection of FAQs tailored for academic researchers working with EXPA1 antibodies, organized by complexity and grounded in experimental methodology. The content integrates principles from antibody engineering, validation, and application in research contexts.
Advanced Research Questions
How can conflicting EXPA1 localization data between IHC and GFP-tagged lines be resolved?
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Troubleshooting workflow:
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Case study: A 2023 study resolved discrepancies by using in situ proximity ligation assays (PLA) to confirm membrane-associated EXPA1 clusters .
What strategies improve EXPA1 antibody efficacy in multiplexed imaging?
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Multiplex optimization:
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Isotype/subtype diversity: Pair EXPA1 (mouse IgG1) with secondary antibodies conjugated to distinct fluorophores (e.g., Alexa Fluor 488 anti-mouse IgG1) and other targets (e.g., Cellulose Synthase, rabbit IgG) .
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Spectral unmixing: Use narrowband filters and reference spectra to minimize bleed-through.
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Validation: Confirm co-localization via confocal Z-stack analysis and line-scan intensity plots .
How do glycosylation states affect EXPA1 antibody binding in planta?
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Experimental design:
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Deglycosylation: Treat plant extracts with PNGase F and compare Western blot band shifts.
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Glycoengineered lines: Use Arabidopsis mutants (e.g., cgl1) with altered N-glycosylation pathways.
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SPR analysis: Measure binding kinetics (KD) of EXPA1 antibody to glycosylated vs. deglycosylated recombinant protein .
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Data Contradiction Analysis Table
| Conflict | Potential Causes | Resolution Strategy |
|---|---|---|
| Varied Western blot bands | Proteolytic cleavage, splice isoforms | Use protease inhibitors; screen isoform-specific primers |
| Inconsistent IHC staining | Fixation method, antibody dilution | Titrate antibody; test multiple retrieval buffers |
| Discrepant functional data | Off-target effects, batch variability | Validate with orthogonal assays (e.g., RNAi) |
