AHL20 Antibody
Description
Definition and Biological Role of AHL20
AHL20 (AT-HOOK MOTIF NUCLEAR LOCALIZED 20) is a member of the AHL protein family in Arabidopsis thaliana. These proteins contain AT-hook DNA-binding motifs and a conserved plant and prokaryote conserved (PPC) domain, enabling interactions with chromatin and transcriptional regulators . AHL20 specifically modulates hypocotyl elongation and flowering time by repressing genes like FLOWERING LOCUS T (FT) and downstream targets (AGL8, SPL3, TSF) .
Key Findings
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Flowering Regulation: Overexpression of AtAHL20 delays flowering by suppressing FT expression, independent of photoperiod .
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Protein Interactions: AHL20 forms homo- and heterotrimers with other AHLs (e.g., AHL19, AHL22, AHL29) via its PPC domain, influencing chromatin structure and gene regulation .
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Structural Requirements: The R-G-R motif in the AT-hook domain is critical for DNA binding and functional activity .
Table 1: AHL20 Functional Characteristics
| Property | Description | Source |
|---|---|---|
| Gene ID | AT3G60840 | |
| Protein Domains | AT-hook motif, PPC domain | |
| Cellular Localization | Nucleus | |
| Mutant Phenotype | Early flowering in AHL20m mutants; delayed flowering in overexpressors |
Antibody Development for AHL Proteins
While the provided sources do not explicitly describe an AHL20-specific antibody, research on related AHL proteins highlights methodologies for antibody generation:
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Polyclonal vs. Monoclonal Antibodies: AHL studies often use polyclonal antibodies for broad epitope recognition or monoclonal antibodies for specificity .
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Validation: Antibodies targeting AHLs are validated via Western blot, immunoprecipitation, and immunohistochemistry (e.g., SOB3/AHL29 antibody) .
Research Applications of AHL Antibodies
Antibodies against AHLs enable:
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Chromatin Interaction Studies: Mapping DNA-protein complexes (e.g., AHL22 binding to FT promoter) .
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Phenotypic Analysis: Tracking protein localization during development (e.g., hypocotyl elongation) .
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Mechanistic Insights: Identifying post-translational modifications or interaction partners .
Table 2: Example Antibody Validation Parameters
Gaps and Future Directions
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Here’s a structured collection of FAQs tailored to academic research on AHL20 Antibody, incorporating methodological guidance and data-driven insights:
Advanced Research Questions
How to resolve contradictions in AHL20 antibody performance across ELISA, Western blot, and ChIP-seq assays?
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Troubleshooting Framework:
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Epitope accessibility: Test antibody binding to denatured (Western) vs. native (ChIP) protein conformations.
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Cross-reactivity screening: Use peptide arrays to identify off-target epitopes in related AT-hook proteins (e.g., AHL23, AHL27) .
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Quantitative correlation: Compare signal intensities across assays using serial dilutions of recombinant AHL20.
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What strategies optimize AHL20 antibody-based chromatin profiling in low-abundance tissue samples?
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Protocol Optimization:
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Combine CUT&Tag with signal amplification systems (e.g., TSA-plus) for low-input chromatin (<10,000 cells).
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Validate genome-wide targets by cross-referencing ChIP-seq peaks with AHL20-binding motif (AT-rich sequences) predictions.
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Use spike-in controls (e.g., Drosophila chromatin) for quantitative normalization.
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Data Integration & Computational Challenges
How to reconcile transcriptomic and proteomic data discrepancies in AHL20 functional studies?
| Data Type | Common Discrepancies | Resolution Strategy |
|---|---|---|
| mRNA-seq | High AHL20 expression | Check for post-transcriptional regulation via miRNA target scans |
| Proteomics | Undetectable protein | Optimize antibody concentration; test alternative extraction buffers |
| Phenotyping | Weak mutant phenotype | Perform combinatorial knockouts with AHL paralogs |
Can machine learning predict AHL20 antibody cross-reactivity across plant species?
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Pipeline:
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Train a CNN model on AHL20 epitope sequences and orthologs from >50 plant species.
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Validate predictions via in silico docking simulations against predicted 3D structures.
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Prioritize experimental testing in species with >85% sequence homology to Arabidopsis AHL20.
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Experimental Design Table
Key parameters for AHL20 antibody applications in Arabidopsis:
| Application | Recommended Dilution | Critical Controls | Validation Metrics |
|---|---|---|---|
| Western Blotting | 1:1,000 | Knockout line, pre-immune serum | Single band at ~25 kDa |
| Immunofluorescence | 1:200 | Secondary-only, tissue-specific KO | Nuclear localization pattern |
| ChIP-seq | 5 µg/IP | IgG control, input DNA normalization | Enrichment at AT-rich promoters |
