AHK1 Antibody
Description
Definition and Scope of AHK1 Antibodies
AHK1 antibodies target the AHK1 protein, which exists in distinct biological contexts:
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Yeast AHK1: A scaffold protein in Saccharomyces cerevisiae that regulates the Hog1 mitogen-activated protein kinase (MAPK) pathway during osmostress .
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Plant AHK1: A histidine kinase in Arabidopsis thaliana that functions as an osmosensor and mediates drought stress responses .
These antibodies enable detection, localization, and functional analysis of AHK1 in experimental settings.
Biological Functions of AHK1
In yeast, AHK1 serves as a scaffold protein that coordinates signaling components:
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Binding Partners: AHK1 interacts with Hkr1 (a putative osmosensor), Sho1, Ste11 (MAPKKK), and Pbs2 (MAPKK) to facilitate Hog1 MAPK activation under osmotic stress .
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Cross-Talk Inhibition: AHK1 prevents signal leakage between the Hog1 and Kss1 MAPK pathways, ensuring signaling fidelity .
In plants, AHK1 is critical for stress adaptation:
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Osmotic Stress Response: AHK1 acts as a positive regulator of drought and osmotic stress signaling, influencing stomatal closure and water retention .
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Homomerization: AHK1 forms homomers, a process essential for its function in stress signaling .
Yeast AHK1 Studies
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Structural Insights:
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Functional Validation:
Plant AHK1 Studies
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Expression and Purification:
Applications of AHK1 Antibodies
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Mechanistic Studies: Used to map AHK1 interactions in yeast signaling complexes .
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Stress Response Research: Facilitate analysis of AHK1’s role in plant drought tolerance .
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Structural Biology: Aid in crystallization efforts to resolve AHK1’s 3D structure .
Comparative Analysis Across Species
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Functional Conservation: Both yeast and plant AHK1 are integral to osmotic stress responses but operate via distinct mechanisms.
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Divergence: Yeast AHK1 lacks kinase activity, whereas plant AHK1 functions as a histidine kinase .
Table 1: Key Studies on AHK1 Antibodies
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Studies demonstrate that de novo abscisic acid biosynthesis, up-regulated through the rate-limiting NCED3 gene, can be triggered by changes in leaf turgor pressure; however, AHK1 does not appear to function as the primary turgor-sensing receptor. (PMID: 28449122)
- Findings suggest that the unimpaired growth, ABA levels, proline accumulation, and solute accumulation observed in *ahk1* mutants under low water potential indicate that AHK1 may not be the principal plant osmosensor for low water potential tolerance. (PMID: 23184230)
- AHK1 plays a significant role in plant growth, as evidenced by the further reduced growth observed in the *ahk1 ahk2 ahk3* triple mutant. (PMID: 18077346)
- The increased water stress tolerance observed upon ATHK1 overexpression suggests a novel mechanism for enhancing drought resistance through receptor-mediated improvements in water status sensing. (PMID: 18441212)
KEGG: ath:AT2G17820
STRING: 3702.AT2G17820.1
Q&A
AHK1 Antibody: Frequently Asked Questions for Academic Researchers
Advanced Research Questions
How can conflicting data on AHK1’s role in MAPK cross-talk be resolved?
Discrepancies often arise from:
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Strain-specific effects: AHK1 disruption in ssk2/22Δ msb2Δ backgrounds reduces Hog1 activation by 50%, while other backgrounds show full pathway redundancy .
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Experimental conditions: Osmotic stress duration impacts Kss1/Hog1 cross-talk detection. Optimize time-course assays with anti-phospho-p38/p44 antibodies .
Example Data Contradiction Resolution:
| Observation | Hypothesis | Validation Method |
|---|---|---|
| AHK1 binds Sho1 but not Msb2 | SH3 domain specificity | SH3-domain truncation mutants (e.g., Sho1-ΔSH3) in co-IP |
What strategies improve AHK1 detection in membrane protein complexes?
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Solubilization: Use 1% digitonin or DDM for native extraction, as demonstrated in yeast membrane fractionation .
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Chromatography: Combine Ni-NTA IMAC (for His-tagged AHK1) with size-exclusion chromatography to isolate monodisperse complexes (~111 kDa) .
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Detergent optimization: 0.015% n-dodecyl-β-D-maltoside (DDM) minimizes aggregation while preserving activity .
How can researchers design experiments to dissect AHK1’s scaffolding vs. enzymatic roles?
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Mutagenesis: Introduce Pro→Ser substitutions in Ahk1’s Sho1-binding motif (residues 849–857) to disrupt scaffolding without affecting kinase activity .
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Kinase-dead mutants: Compare signaling outcomes (e.g., Hog1 phosphorylation) in AHK1 kinase-dead vs. scaffold-disrupted strains.
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Biophysical assays: Use CD spectroscopy to confirm structural integrity of mutants (α-helix content ≥45%) .
Technical Optimization
What are common pitfalls in quantifying AHK1-dependent Hog1 activation?
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Signal saturation: Use diluted lysates and anti-phospho-p38 antibodies with enhanced chemiluminescence (ECL) detection .
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Non-linear quantification: Calibrate band intensities against recombinant phospho-Hog1 standards.
Troubleshooting Table:
| Issue | Cause | Solution |
|---|---|---|
| Weak AHK1 signal | Low expression | Induce with 0.4 mM CuSO₄ for 6–8 hr |
| Cross-reactive bands | Incomplete blocking | Use 5% BSA + 0.1% Tween-20 in TBST |
