At4g31480 Antibody
Description
Biological Context of Target Protein
The At4g31480 gene encodes the β-subunit of the COPI (coatomer protein complex I), which mediates retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum. Key functional insights include:
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Genetic Diversity: Analysis of 1,135 Arabidopsis accessions revealed moderate nucleotide diversity (π = 0.0015) for At4g31480, with single-nucleotide polymorphisms (SNPs) showing non-synonymous substitutions predicted to alter protein function .
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Cellular Role: β-COP is critical for maintaining Golgi structure and facilitating protein sorting. Silencing At4g31480 via amiRNA resulted in dwarf phenotypes, reduced seed production, and developmental defects in Arabidopsis .
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Iron Uptake: Proteomic studies identified β-COP as part of a root epidermal protein complex regulating high-affinity iron uptake, highlighting its role in nutrient homeostasis .
3.1. Functional Studies
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Mutant Phenotypes: Transgenic Arabidopsis lines with At4g31480 silencing exhibited:
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Protein Interactions: β-COP associates with dynamin-related proteins (e.g., DRP3A) and clathrin adaptors, suggesting coordination between COPI vesicles and endocytic pathways .
3.2. Comparative Proteomics
A mass spectrometry analysis of Arabidopsis root proteins identified β-COP among intracellular trafficking components:
| Category | Accession | Protein Name | Unique Peptides Detected |
|---|---|---|---|
| Intracellular Trafficking | AT4G31480 | Coatomer β subunit (COPI) | 2 |
| AT1G62020 | Coatomer α subunit (COPI) | 2 | |
| AT3G63460 | SEC31b (COPII component) | 6 |
This table underscores β-COP’s role in vesicle transport networks .
Technical Considerations
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Antibody Validation: The antibody has been validated for Arabidopsis-specific reactivity in WB and ELISA, with no cross-reactivity reported in other plant species .
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Epitope Stability: The immunogen sequence can be customized upon request, enabling studies of specific β-COP domains or post-translational modifications .
Future Directions
Current research gaps include:
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Structural characterization of Arabidopsis β-COP using cryo-EM.
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Role of COPI in stress responses (e.g., pathogen infection, abiotic stress).
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Development of β-COP knockout lines to dissect trafficking pathways in planta.
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
Experimental Design for At4g31480 Antibody Validation
Question: How can researchers validate the specificity and sensitivity of the At4g31480 antibody in experimental settings?
Answer: Validation of the At4g31480 antibody involves several steps:
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Western Blot (WB): Use WB to confirm the antibody's ability to bind specifically to the At4g31480 protein in Arabidopsis extracts. Include controls such as pre-immune serum or a secondary antibody alone to assess background signal.
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ELISA: Employ ELISA to quantify the antibody's binding affinity. This involves coating plates with recombinant At4g31480 protein and measuring optical density (OD) values at different antibody dilutions.
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Immunolocalization: Perform immunofluorescence or immunohistochemistry to visualize the protein's localization within plant tissues.
Data Analysis and Contradiction Resolution
Question: How can researchers resolve discrepancies in data obtained from different assays using the At4g31480 antibody?
Answer: Discrepancies often arise due to differences in assay sensitivity, specificity, or sample preparation. To resolve these:
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Standardize Assay Conditions: Ensure consistent conditions across all experiments, including buffer composition, temperature, and antibody concentrations.
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Use Controls: Include positive and negative controls in each assay to validate results.
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Statistical Analysis: Apply appropriate statistical tests to compare results from different assays, considering factors like sample size and variability.
Advanced Research Questions: Antibody Optimization
Question: What strategies can researchers use to optimize the At4g31480 antibody for improved specificity or affinity?
Answer: Optimization strategies include:
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Affinity Maturation: Use techniques like phage display or yeast display to select for higher affinity variants of the antibody.
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Epitope Mapping: Identify the specific epitope recognized by the antibody to improve specificity or design blocking peptides for competition assays.
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Antibody Engineering: Employ computational tools or mutagenesis to modify the antibody's structure, enhancing its binding properties.
Methodological Considerations for At4g31480 Antibody Applications
Question: What methodological considerations are crucial when applying the At4g31480 antibody in various research applications?
Answer: Key considerations include:
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Sample Preparation: Ensure proper extraction and purification of proteins from Arabidopsis tissues to minimize background and enhance signal.
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Antibody Concentration: Optimize antibody dilutions for each assay to achieve the best signal-to-noise ratio.
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Cross-reactivity: Assess potential cross-reactivity with other Arabidopsis proteins to confirm specificity.
Interpretation of Results in the Context of Plant Biology
Question: How can researchers interpret the results from studies using the At4g31480 antibody in the broader context of plant biology?
Answer: Interpretation involves understanding the role of the At4g31480 protein in plant cellular processes. This includes:
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Functional Analysis: Relate the protein's expression patterns to specific physiological or developmental processes in plants.
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Comparative Studies: Compare findings across different plant species or conditions to identify conserved functions.
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Integration with Other Data: Combine antibody data with other molecular or biochemical analyses to provide a comprehensive view of plant biology.
Example Data Table for ELISA Analysis
| Dilution Factor | Optical Density (OD) |
|---|---|
| 1:100 | 0.8 |
| 1:500 | 0.4 |
| 1:1000 | 0.2 |
| Negative Control | 0.05 |
This table illustrates how ELISA data can be used to assess the binding affinity of the At4g31480 antibody by measuring optical density at different dilutions.
Detailed Research Findings
The At4g31480 antibody targets a protein involved in the early secretory pathway of Arabidopsis, specifically related to COPI-coated vesicles. Research using this antibody can provide insights into plant cell trafficking mechanisms and their implications for plant development and stress responses.
