At4g23580 Antibody
Description
Definition and Target Identification
The At4g23580 antibody is a polyclonal or monoclonal antibody specifically designed to detect the protein product of the Arabidopsis thaliana gene At4g23580, which encodes a PAS domain-containing protein tyrosine kinase family protein . This gene is part of the RAF-related mitogen-activated protein kinase kinase kinase (MAPKKK) family, classified under subgroup B2 in Arabidopsis .
Key Features of the At4g23580 Protein:
Antibody Development and Validation
The At4g23580 antibody is typically generated using recombinant protein fragments or synthetic peptides corresponding to the target sequence. Commercial versions (e.g., PHY1430A) are lyophilized for stability and require reconstitution in PBS or similar buffers .
Role in Plant Stress Responses
Studies using the At4g23580 antibody have linked its target protein to abiotic stress signaling. For example:
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Oxidative Stress: The PAS domain facilitates redox sensing, modulating kinase activity under stress .
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Light Signaling: Interacts with photoreceptors to regulate photomorphogenesis .
Genetic Interactions
At4g23580 is co-expressed with genes involved in auxin signaling and brassinosteroid biosynthesis . Key interactors include:
| Gene ID | Function | Interaction Evidence |
|---|---|---|
| AT2G41000 | Chaperone protein (stress response) | Yeast two-hybrid assays . |
| AT5G57150 | Basic helix-loop-helix transcription factor | Co-expression networks . |
Validation and Challenges
Antibody specificity is critical for reproducibility. The At4g23580 antibody has been validated via:
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Knockout Controls: Loss of signal in At4g23580 mutant lines .
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Cross-Reactivity Tests: No off-target binding observed in related MAPKKKs .
Significance in Plant Biology
The At4g23580 antibody enables researchers to:
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Map kinase signaling pathways in environmental adaptation.
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Study crosstalk between hormonal and stress-responsive networks .
For future studies, combining this antibody with CRISPR-edited mutants could clarify its role in crop resilience .
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Q&A
Here’s a structured collection of FAQs for researchers working with the AT4G23580 antibody, designed to address both foundational and advanced research challenges while incorporating methodologies from cutting-edge studies:
How to validate the specificity of the AT4G23580 antibody in plant protein extracts?
Methodology:
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Perform Western blotting using plant tissue lysates from wild-type and AT4G23580 knockout mutants. Compare band patterns to confirm absence of signal in knockouts .
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Use immunofluorescence with confocal microscopy to verify subcellular localization (e.g., endosperm-specific expression) .
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Validate via peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm loss of signal .
What experimental controls are critical for AT4G23580 antibody-based assays?
Key Controls:
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Negative controls: Knockout mutants or siRNA-treated samples .
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Cross-reactivity checks: Test against homologs (e.g., MIPS1, MIPS2, MIPS3) using purified proteins .
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Buffer compatibility: Compare signal stability in native vs. denaturing conditions (e.g., using urea or SDS) .
How to resolve contradictions in AT4G23580 localization data across studies?
Approach:
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Conduct multi-method validation:
Method Strength Limitation Subcellular fractionation High purity Risk of organelle cross-contamination Immunogold EM Nanoscale resolution Low throughput Live-cell imaging Dynamic tracking Requires transgenic lines -
Compare antibody performance with orthogonal tools (e.g., GFP-tagged AT4G23580) .
What computational tools can predict AT4G23580 epitopes for antibody optimization?
Framework:
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Use residue-level energy decomposition (as in ABDPO ) to model antibody-antigen interactions.
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Apply molecular dynamics simulations to assess binding stability under physiological conditions .
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Validate predictions via alanine-scanning mutagenesis of candidate epitopes .
How to design a study investigating AT4G23580’s role in stress responses?
Protocol:
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Phenotypic screening: Expose AT4G23580 mutants to abiotic stresses (drought, salinity).
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Multi-omics integration:
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Transcriptomics: RNA-seq under stress conditions.
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Interactomics: Co-IP/MS to identify binding partners.
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Functional validation: CRISPR-edited lines complemented with epitope-tagged variants .
Conflicting reports on AT4G23580’s enzymatic activity – how to reconcile?
Resolution Strategy:
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Activity assays: Compare galactose oxidase activity in recombinant protein vs. native extracts .
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Post-translational modification (PTM) analysis: Use middle-down MS to detect phosphorylation or glycosylation .
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Structural studies: Cryo-EM or X-ray crystallography to identify regulatory domains .
