At5g56430 Antibody
Description
Overview of At5g56430 Antibody
The At5g56430 antibody is a polyclonal reagent developed for research applications targeting the Arabidopsis thaliana protein encoded by the At5g56430 gene. This antibody is primarily utilized in plant molecular biology to study the functional role of the At5g56430 protein, which is implicated in stress response pathways and phosphorelay signaling .
Biological Role of At5g56430 Protein
The At5g56430 protein is a histidine kinase homolog involved in:
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Stress Response: Regulates hypocotyl elongation under osmotic stress .
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Phosphoproteome Dynamics: Modulates phosphorylation cascades during mannitol-induced stress, transitioning from His-to-Asp phosphorelay to Ser/Thr/Tyr phosphorylation .
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Pathogen Interaction: Implicated in responses to fungal pathogens like Alternaria brassicicola .
4.1. Mechanistic Studies
The antibody has been employed to investigate:
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AHK1-ED Interactions: The extracellular domain (ED) of AHK1 (At5g56430) interacts with BAK1, a regulator of brassinosteroid signaling, under etiolated growth conditions .
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Phosphorylation Networks: Comparative phosphoproteomics of ahk1 mutants revealed altered phosphorylation patterns in cytoskeleton-associated proteins and transcription factors .
4.2. Validation and Limitations
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Specificity: Validated using KO cell lines and adsorption controls, aligning with best practices for antibody reliability .
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Limitations: Requires stringent validation in non-model plant species due to potential cross-reactivity with homologous proteins .
Comparative Analysis of Antibody Performance
| Feature | At5g56430 Antibody | Typical Polyclonal Antibodies |
|---|---|---|
| Target Specificity | High (validated in Arabidopsis) | Variable (species-dependent) |
| Lead Time | 14–16 weeks | 4–6 weeks (pre-validated stocks) |
| Applications | ELISA, WB | WB, IHC, IF |
| Storage Stability | Stable at -80°C | Often requires lyophilization |
Key Research Findings
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Stress Adaptation: At5g56430-deficient Arabidopsis lines exhibit hypersensitivity to osmotic stress, highlighting its role in drought tolerance .
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Pathogen Response: AHK1-dependent signaling enhances resistance to necrotrophic fungi, suggesting biotechnological potential for crop improvement .
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Structural Insights: Homology modeling of AHK1-ED challenges prior hypotheses about mechanosensitive signal perception, prompting re-evaluation of ligand-binding mechanisms .
Future Directions
Product Specs
Q&A
FAQs for Researchers on At5g56430 Antibody in Academic Research
How to validate At5g56430 antibody specificity in Arabidopsis thaliana tissues?
Methodological Answer:
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Western Blot with Knockout Mutants: Use Arabidopsis T-DNA insertion lines lacking functional At5g56430 (AHK1) as negative controls. Compare protein bands in wild-type vs. mutant extracts .
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Immunoprecipitation (IP) Followed by Mass Spectrometry: Perform IP using the antibody and verify co-purified proteins via MS to confirm target specificity .
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Cross-Reactivity Testing: Test against recombinant proteins of closely related histidine kinases (e.g., AHK2, AHK3) to rule out nonspecific binding .
Table 1: Key Validation Metrics
| Assay | Expected Outcome (Wild-Type) | Negative Control (Mutant) |
|---|---|---|
| Western Blot | ~130 kDa band | No band |
| Immunofluorescence | Membrane-associated signal | Cytosolic/no signal |
| ELISA (Recombinant) | High OD450 (>2.0) | Low OD450 (<0.5) |
What are common experimental applications of At5g56430 antibodies in plant signaling studies?
Methodological Answer:
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Subcellular Localization: Use confocal microscopy with GFP-tagged lines or immunofluorescence in fixed root tissues .
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Protein-Protein Interaction: Deploy co-IP with known interactors (e.g., AHP1, AHP2) under osmotic stress conditions .
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Quantitative Analysis: Combine ELISA with tissue-specific promoters to measure AHK1 expression gradients in response to cytokinin .
How to design experiments resolving contradictory data on AHK1’s role in abscisic acid (ABA) signaling?
Methodological Answer:
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Context-Specific KO Lines: Generate conditional knockout mutants (e.g., ethanol-inducible Cre-lox) to study tissue-specific effects .
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Phosphorylation-State-Specific Antibodies: Develop or source antibodies targeting phosphorylated His residues in AHK1’s kinase domain to differentiate activation states .
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Multi-Omics Integration: Correlate AHK1 protein levels (via antibody-based assays) with transcriptomic data from ABA-treated vs. untreated plants .
Table 2: Resolving Data Contradictions
| Conflict Source | Proposed Resolution Strategy | Key Controls |
|---|---|---|
| Tissue-specific roles | Cell-type-specific promoter-driven RNAi | Whole-plant phenotyping |
| Environmental crosstalk | Combinatorial stress treatments (e.g., ABA + drought) | Hormone quantification |
What advanced methods address low antibody specificity in detecting post-translational modifications (PTMs) of AHK1?
Methodological Answer:
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Phos-Tag SDS-PAGE: Resolve phosphorylated vs. non-phosphorylated AHK1 isoforms using phosphate-binding tags .
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Ubiquitination Assays: Combine IP with anti-ubiquitin antibodies under proteasome inhibition (MG132 treatment) .
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Structural Epitope Mapping: Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify antibody-binding regions affected by PTMs .
How to optimize ChIP-seq protocols for studying AHK1’s transcriptional regulatory role?
Methodological Answer:
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Crosslinking Optimization: Test formaldehyde (1%) vs. dual crosslinkers (e.g., DSG + formaldehyde) for chromatin fixation .
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Sonication Efficiency: Fragment chromatin to 200–500 bp and verify via agarose gel electrophoresis .
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Data Validation: Use qPCR on immunoprecipitated DNA for known AHK1-binding loci (e.g., ARR5 promoter) .
Discrepancies in AHK1 localization under salt stress: Membrane vs. nuclear signals
Resolution Strategy:
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Fractionation-Western Blot: Separate membrane, cytosolic, and nuclear fractions from salt-stressed plants .
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Time-Course Imaging: Track subcellular relocalization at 0, 15, 60, and 120 minutes post-stress.
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Competitive Peptide Blocking: Pre-incubate antibody with excess AHK1 peptide to confirm signal specificity .
Critical Controls:
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Marker Proteins: Include membrane (PIP2A) and nuclear (HISTONE H3) markers in fractionation assays.
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Alternative Antibodies: Compare results with independently validated AHK1 antibodies .
High-throughput antibody validation using NGS-coupled platforms
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NGS-Based Clonal Screening: Use single-cell sequencing to link antibody binding to B-cell receptor sequences, ensuring epitope diversity coverage .
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Automated Epitope Binning: Deploy SPR (Surface Plasmon Resonance) with overlapping AHK1 peptide arrays to map binding sites .
Table 3: NGS Data Analysis Workflow
| Step | Tool/Platform | Output Metric |
|---|---|---|
| Raw Sequence QC | Geneious Biologics | Read quality (Q-score >30) |
| V(D)J Annotation | IgBLAST | Germline alignment score |
| Clonal Diversity | Shannon Index | Clonotype richness |
