AIG2LB Antibody
Description
Molecular Characterization of AIG2LB
AIG2LB belongs to the AIG2 (AvrRpt2-Induced Gene 2) family, which includes paralogs such as AIG2LA and AIG2B. Key features include:
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Sequence Identity: AIG2LB shares only 17% amino acid identity with its human orthologue, suggesting a specialized role in plant systems .
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Domain Structure: Contains putative γ-glutamyl cyclotransferase (GGCT) catalytic sites critical for enzymatic activity and immune signaling .
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Localization: Co-localizes with biosynthetic enzymes for tryptophan-derived secondary metabolites (TDSMs), indicating metabolic involvement .
Functional Role in Plant Immunity
AIG2LB and its homologs fine-tune defense systems by balancing salicylic acid (SA) and TDSM pathways:
Key Mechanisms
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Gatekeeper Function: Prevents cross-activation between SA-mediated defenses and TDSM chemical responses, ensuring balanced immune output .
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Pathogen Response: Gene expression is induced by pathogen-associated molecular patterns (e.g., flg22, chitooligosaccharides) and virulent bacterial strains like Pseudomonas syringae .
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ABA Signaling: Knockdown experiments (via amiRNA) show reduced ABA-mediated gene induction (e.g., RAB18), linking AIG2LB to abiotic stress responses .
Table 1: AIG2 Family Functional Comparisons
| Protein | Immune Role | Expression Triggers | Mutant Phenotype |
|---|---|---|---|
| AIG2LB | Modulates ABA and TDSM pathways | Pathogen elicitors, ABA | Reduced ABA signaling |
| AIG2A | Limits SA activation by TDSMs | SA, bacterial pathogens | Enhanced pathogen resistance |
| AIG2B | Co-regulates SA-TDSM balance | General pathogen signals | Hyperactive SA defenses |
Antibody Development and Applications
While no commercial AIG2LB-specific antibody is documented, antibody engineering principles (e.g., structure-function relationships) provide a framework for its potential development:
Antibody Design Considerations
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Epitope Targeting: Prioritize regions with low homology to human GGCTs (e.g., catalytic sites) to avoid cross-reactivity .
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Validation: Use plant models (e.g., Arabidopsis mutants) to confirm specificity and functional blocking .
Table 2: Antibody Performance Metrics (Hypothetical)
| Parameter | Specification |
|---|---|
| Host Species | Rabbit |
| Immunogen | Recombinant AIG2LB catalytic domain |
| Application | Western blot, immunoprecipitation |
| Cross-Reactivity | None with AIG2LA or human GGCTs |
Research Gaps and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
KEGG: ath:AT5G39730
UniGene: At.9359
Q&A
Given the specific nature of the "AIG2LB Antibody" and its relevance to academic research, particularly in plant biology, here is a collection of FAQs tailored to researchers. These questions and answers aim to address both basic and advanced research scenarios, focusing on experimental design and data analysis.
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To study the role of AIG2LB in plant defense, you can use the AIG2LB antibody in immunoblotting or immunolocalization assays. First, prepare plant samples under different conditions (e.g., pathogen-infected vs. non-infected). Then, use the AIG2LB antibody to detect changes in AIG2LB protein levels or localization. This will help understand how AIG2LB is involved in plant defense responses.
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When interpreting contradictory results, consider the following:
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Experimental Conditions: Differences in plant species, growth conditions, or pathogen types might affect AIG2LB expression.
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Antibody Specificity: Ensure the AIG2LB antibody is specific and does not cross-react with other proteins.
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Methodological Variations: Techniques like Western blotting or immunofluorescence might yield different results due to sensitivity or specificity issues.
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To study protein-protein interactions involving AIG2LB, you can use co-immunoprecipitation (co-IP) assays. First, express AIG2LB in a suitable system (e.g., Arabidopsis protoplasts). Then, use the AIG2LB antibody to pull down AIG2LB and associated proteins. Analyze the precipitated proteins using mass spectrometry or Western blotting to identify interacting partners.
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To validate the specificity of the AIG2LB antibody:
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Western Blotting: Use the antibody on protein extracts from AIG2LB-expressing and non-expressing plants to check for specific bands.
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Immunolocalization: Perform immunofluorescence or immunohistochemistry to confirm the expected localization pattern of AIG2LB.
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Peptide Competition Assays: Use synthetic peptides corresponding to the AIG2LB epitope to compete with the antibody binding, ensuring specificity.
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For quantitative analysis, use techniques like quantitative Western blotting or immunoprecipitation followed by mass spectrometry. These methods allow for precise measurement of AIG2LB levels under various conditions, such as pathogen infection or hormone treatments.
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To ensure reliability:
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Negative Controls: Include samples without the AIG2LB antibody or with a non-specific antibody.
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Positive Controls: Use known AIG2LB-expressing samples.
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Biological Replicates: Perform experiments on multiple independent biological samples.
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Technical Replicates: Repeat each assay multiple times to account for technical variability.
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For statistical analysis:
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Hypothesis Testing: Use appropriate tests (e.g., t-test, ANOVA) to compare AIG2LB levels between conditions.
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Data Normalization: Normalize data to account for loading variations in Western blots.
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Error Bars and Confidence Intervals: Include these in graphs to visualize variability and significance.
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To study the functional role of AIG2LB, you can use RNA interference (RNAi) or CRISPR-Cas9 to knockdown or knockout AIG2LB in plants. Then, use the AIG2LB antibody to confirm the reduction in AIG2LB levels. Analyze the resulting phenotypes and defense responses to pathogens to understand AIG2LB's role.
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For optimal sample preparation:
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Tissue Homogenization: Use gentle homogenization methods to preserve protein integrity.
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Buffer Selection: Choose buffers that minimize protein degradation and preserve epitope recognition.
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Protein Extraction: Optimize extraction conditions to maximize AIG2LB recovery.
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To study subcellular localization, perform immunofluorescence microscopy using the AIG2LB antibody. This involves fixing and permeabilizing plant cells, followed by incubation with the primary antibody and a fluorescently labeled secondary antibody. Visualize the localization pattern using a confocal microscope.
Example Data Table: Quantitative Analysis of AIG2LB Levels
| Condition | AIG2LB Level (Relative Units) |
|---|---|
| Control | 1.0 ± 0.2 |
| Pathogen | 2.5 ± 0.5 |
| Hormone | 1.8 ± 0.3 |
