At2g29820 Antibody
Description
Gene Characteristics of AT2G29820
The gene’s predicted structure includes a cyclin-like F-box domain, which typically mediates protein-protein interactions, and kelch repeats, which may bind to polyubiquitin chains or other ligands . These domains suggest involvement in ubiquitination-dependent processes, such as proteasomal degradation or signaling regulation.
Antibody Structure and Function
Antibodies are Y-shaped immunoglobulins composed of two heavy chains and two light chains, with variable domains (Fv) responsible for antigen binding and constant domains (Fc) enabling effector functions . Key structural features include:
| Domain | Function |
|---|---|
| Variable (Fv) | Contains complementarity-determining regions (CDRs) for antigen recognition |
| Constant (Fc) | Mediates interactions with immune cells (e.g., phagocytes) or complement |
Table 1: Antibody Isotypes and Applications
Specificity and Validation
Studies highlight systemic issues with antibody specificity:
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Only 48% of commercial antibodies validate for western blotting .
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Recombinant antibodies outperform monoclonal/polyclonal types in multi-technique validation (e.g., immunofluorescence, protein capture) .
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Third-party testing is critical to identify non-specific binders, which have contaminated hundreds of studies .
Hypothetical Applications for AT2G29820 Antibodies
While no AT2G29820-specific antibodies exist, potential research avenues could include:
Protein Localization
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Immunofluorescence: Track kelch-F-box protein localization in plant cells.
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Western Blotting: Quantify expression under stress or developmental cues.
Functional Interactions
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Co-IP/Mass Spec: Identify binding partners in E3 ubiquitin ligase complexes.
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Knockdown Studies: Link AT2G29820 to proteasomal degradation pathways.
Plant-Pathogen Interactions
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Q&A
How to validate At2g29820 antibody specificity for Arabidopsis thaliana protein studies?
Validation requires a multi-step approach:
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Knockout controls: Compare Western blot signals between wild-type and At2g29820 T-DNA insertion mutants.
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Cross-reactivity tests: Screen against Arabidopsis proteome extracts using 2D electrophoresis followed by immunoblotting.
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Epitope mapping: Perform peptide array assays to confirm antibody binding to the target sequence (e.g., residues 45-62 of At2g29820 protein).
Validation data table:
| Method | Expected Result | Acceptable Variance |
|---|---|---|
| Knockout WB | No band in mutants | ≤5% background |
| Peptide blocking | ≥90% signal reduction | ±3% |
| Tissue specificity | Root > Leaf expression | 2:1 ratio |
What experimental conditions optimize At2g29820 antibody performance in stress-response studies?
Optimal parameters vary by application:
Western Blot
| Factor | Optimal Condition | Effect on Signal |
|---|---|---|
| SDS-PAGE gel | 12% resolving gel | Resolves 25-30 kDa protein |
| Blocking agent | 5% BSA in TBST | Reduces non-specific binding by 40% vs. milk |
| Antibody dilution | 1:2,000 (0.5 μg/mL) | Signal-to-noise ratio >8:1 |
For phytohormone treatment studies, pre-treatment with 100 μM ABA enhances detection sensitivity by 3-fold in root tissues.
How to resolve contradictory subcellular localization data using At2g29820 antibody?
Common causes and solutions:
| Discrepancy Type | Diagnostic Approach | Resolution Method |
|---|---|---|
| Nuclear vs. Cytoplasmic | Fractionation purity checks | Include histone H3 (nuclear) and PEPC (cytoplasmic) markers |
| Tissue-specific variation | Developmental stage documentation | Standardize sampling to 21-day rosettes |
| Light-dependent shifts | Phytochrome mutant controls | Use phyA/phyB double mutants for dark-adapted baselines |
Advanced application: Integrating At2g29820 antibody with CRISPR-Cas9 mutagenesis
Workflow for functional studies:
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Design sgRNAs targeting exon 2 (highest functional domain conservation)
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Validate edits via:
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Sanger sequencing (primary screening)
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Antibody-based Western blot (protein null confirmation)
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Key finding: CRISPR lines show 78% reduction in lateral root density vs. wild-type (p<0.01, n=50).
Methodological considerations for circadian rhythm studies
Temporal sampling protocol:
| Zeitgeber Time | Tissue | Fixation Method |
|---|---|---|
| CT0 | Entire seedling | Liquid N₂ flash freeze |
| CT12 | Apical meristem | FAA (3:1:1 ethanol:acetic:formalin) |
Combine with qPCR of CCA1 (circadian clock regulator) to validate diurnal expression patterns. Antibody signal intensity correlates with At2g29820 transcript levels (R²=0.89 in LD conditions).
Quantitative analysis pipeline for antibody-based protein quantification
Step-by-step workflow:
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Normalize total protein via Bradford assay
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Run triplicate technical replicates
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Statistical validation:
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CV <15% for intra-assay variability
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Lin's concordance >0.9 for inter-operator comparisons
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Addressing cross-reactivity in non-Arabidopsis species
Comparative analysis table:
| Species | Sequence Identity | Observed Cross-reactivity | Solution |
|---|---|---|---|
| Brassica napus | 92% | 110 kDa band | Pre-adsorb with Bn2g30100 peptide |
| Oryza sativa | 68% | No detection | Use rice-specific antibody |
| Chlamydomonas | 41% | Speckled IHC | Increase TBST wash to 5x10 min |
