At2g20870 Antibody

Basic Research Questions

How to validate At2g20870 antibody specificity in Arabidopsis abiotic stress studies?

Validation requires a multi-step approach:

• Knockout controls: Use At2g20870 knockout lines (e.g., T-DNA mutants) to confirm absence of signal in Western blots or immunolocalization .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (e.g., residues 50-150 of At2g20870) to block binding .

• Cross-species testing: Verify reactivity against orthologs in Nicotiana or other model plants to assess conservation .

What are standard applications for At2g20870 antibody in ABA signaling research?

• Protein localization: Immunolocalization in root/shoot apical meristems under drought stress (e.g., paraffin-embedded sections) .

• Quantitative assays: Measure ABA accumulation in transgenic lines overexpressing At2g20870 (e.g., 35S::XERICO) via ELISA, correlating with drought tolerance phenotypes .

• Interaction studies: Co-immunoprecipitation with partners like AtUBC8 or AtTLP9, validated via yeast two-hybrid assays .

How to optimize Western blot protocols for At2g20870 detection?

• Sample preparation: Use fresh tissue homogenized in RIPA buffer with protease inhibitors (e.g., PMSF).

• Membrane blocking: 5% non-fat milk in TBST for 1 hr to reduce non-specific binding .

• Antibody dilution: Start at 1:1,000 for primary antibody (empirically adjust based on signal-to-noise ratio) .

Advanced Research Questions

How to resolve discrepancies in At2g20870 expression data across drought stress studies?

What mechanistic insights can be gained from At2g20870 interaction studies?

• Ubiquitination pathways: At2g20870’s RING-H2 domain interacts with E2 ligases (e.g., AtUBC8), suggesting a role in protein turnover under stress .

• Transcriptional regulation: Co-expression with 9-cis-epoxycarotenoid dioxygenase (NCED3) and RD29a implies ABA biosynthesis modulation .

• Structural analysis: Epitope mapping (e.g., residues 40-60) reveals binding sites critical for enzymatic inhibition or activation .

How to address cross-reactivity with paralogs like At1g56430 in Brassicales?

• Phylogenetic alignment: Identify conserved regions (e.g., RING-H2 motif) and design blocking peptides for unique epitopes .

• CRISPR-Cas9 knockdown: Generate At1g56430 mutants to isolate At2g20870-specific signals .

• Alternative detection: Combine antibody data with RNA-seq to confirm transcriptional vs. post-translational regulation .

Methodological Best Practices

Critical controls for immunoprecipitation (IP) experiments

• Isotype control: Use non-specific IgG to exclude background binding .

• Input normalization: Load 10% of lysate pre-IP to confirm target presence .

• Competition validation: Include excess immunizing peptide (10x molar ratio) to abolish signal .

Quantitative analysis of At2g20870-mediated ABA accumulation

• Calibration curve: Use synthetic ABA (0–100 ng/mL) for ELISA standardization .

• Biological replicates: Three independent T3 homozygous lines (e.g., SS8-3) to account for transgene positional effects .

• Statistical thresholds: Report mean ± S.D. (n=3) with p<0.001 for drought tolerance assays .

Data Interpretation Guidelines

Distinguishing artifact vs. true signal in immunohistochemistry

• False positives: Autofluorescence in vascular tissues; counterstain with DAPI and use confocal microscopy .

• Thresholding: Define positivity as ≥3-fold intensity over WT in ImageJ analyses .

Validating antibody specificity for high-throughput screens

• Multiplex validation: Pair antibody data with orthogonal methods (e.g., RT-qPCR for At2g20870 mRNA) .

• Machine learning: Train classifiers on knockout vs. WT datasets to filter non-specific signals .