IAA28 Antibody
Description
Introduction to IAA28 Protein and Antibody Development
IAA28 (Indoleacetic Acid-Induced Protein 28) is a member of the Arabidopsis Aux/IAA gene family, functioning as a transcriptional repressor in auxin signaling pathways . It regulates lateral root initiation and hypocotyl elongation by modulating auxin-responsive gene expression . The IAA28 antibody is a specialized tool developed to study this protein's localization, interaction partners, and regulatory mechanisms in plant development.
Key Applications of IAA28 Antibody in Plant Research
The IAA28 antibody enables precise detection and characterization of IAA28 protein dynamics. Key applications include:
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Western Blot: Quantifying IAA28 protein levels in root and stem tissues under auxin treatment .
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Immunoprecipitation (IP): Isolating IAA28 complexes to study interactions with auxin response factors (ARFs) like ARF7 and ARF19 .
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Chromatin Immunoprecipitation (ChIP): Mapping IAA28 binding sites on promoters of auxin-responsive genes (e.g., LBD16, LBD29) .
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Functional Studies: Validating gain-of-function mutations (e.g., iaa28-1 P53L substitution) that confer auxin resistance .
Molecular Characterization
IAA28 contains four conserved domains (I–IV), with domain II critical for protein stability. The iaa28-1 mutation (P53L in domain II) stabilizes the protein, leading to auxin resistance and lateral root defects .
Table 1: Key Mutations in Aux/IAA Proteins
| Protein | Mutation | Domain | Phenotype | Reference |
|---|---|---|---|---|
| IAA28 | P53L | II | Auxin-resistant roots | |
| IAA3/SHY2 | C→S redox | II | Altered ROS response | |
| IAA7 | G→R revertant | I/III | Partial loss of repression |
Regulatory Mechanisms
IAA28 suppresses lateral root initiation by repressing ARF-activated transcription of LBD16/29 . Its degradation via the 26S proteasome pathway (half-life ~14.8 minutes) is auxin-dependent .
Table 2: Protein Stability Metrics
| Protein | Half-Life (min) | Degradation Pathway | Reference |
|---|---|---|---|
| IAA28 (wild-type) | 14.8 ± 1.4 | 26S proteasome | |
| IAA17 (truncated) | 4.6 ± 0.4 | 26S proteasome |
Challenges and Considerations
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Specificity: Cross-reactivity with other Aux/IAA proteins (e.g., IAA15, IAA19) requires validation via knockout controls .
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Redox Sensitivity: IAA28 multimerization under oxidative stress may alter antibody binding efficiency .
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Epitope Masking: Post-translational modifications (e.g., phosphorylation) in domain II could affect detection .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- miR847 targets the IAA28 mRNA, encoding an auxin/indole acetic acid (Aux/IAA) repressor protein, for cleavage. PMID: 25794935
Q&A
Here’s a structured FAQ collection for researchers working with IAA28 Antibody in plant molecular biology, synthesized from peer-reviewed studies and technical resources:
How is IAA28 antibody validated for specificity in Arabidopsis studies?
Methodological steps include:
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Immunoblot analysis using Arabidopsis wild-type (Col-0) and iaa28 mutant protein extracts to confirm absence of cross-reactivity .
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Complementation assays with transgenic lines expressing epitope-tagged IAA28 (e.g., FLAG-IAA28) to verify antibody recognition .
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Proteasome inhibition (e.g., 10 µM MG132) to stabilize IAA28 protein for detection in degradation assays .
What are common experimental applications of IAA28 antibody?
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Western blotting: Detect IAA28 protein levels in auxin-treated vs. untreated seedlings (e.g., 1 µM IAA for 6–12 hr) .
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Chromatin immunoprecipitation (ChIP): Identify IAA28-bound promoters (e.g., ABI3, LBD16) using anti-FLAG/IAA28 antibody .
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Mutant phenotyping: Compare lateral root formation in iaa28-1 (gain-of-function) and wild-type under auxin induction .
How to select appropriate mutant lines for IAA28 functional studies?
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Use ethyl methanesulfonate (EMS)-induced mutants (e.g., iaa28-1 with Pro53→Leu substitution) .
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Validate mutants via genetic complementation (e.g., reintroducing wild-type IAA28 genomic DNA) .
How to resolve contradictions in IAA28’s role in auxin signaling?
Case study: Conflicting reports on IAA28’s repressor vs. activator roles in lateral root initiation.
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Approach:
How to design a ChIP-seq workflow for IAA28-DNA interaction studies?
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Protocol:
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Key controls:
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IgG-isotype negative control.
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Input DNA normalization.
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What methods optimize IAA28 antibody use in high-throughput screens?
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Multiplexed Western blotting: Pair IAA28 antibody with housekeeping proteins (e.g., Rubisco) for normalization .
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Automated image analysis: Quantify band intensity using tools like ImageJ or Licor Odyssey .
Interpreting conflicting IAA28 antibody signals in immunoblots
| Condition | WT Signal | iaa28-1 Signal | Interpretation |
|---|---|---|---|
| No auxin | Low | Low | Baseline degradation |
| 10 µM IAA | High | Moderate | Partial stabilization |
| IAA + MG132 | Very high | High | Proteasome dependence |
