At4g08875 Antibody

FAQs for At4g08875 Antibody in Arabidopsis thaliana Research

Advanced Research Questions

• What strategies resolve low signal-to-noise ratios in At4g08875 immunoprecipitation (IP) assays?

  • Optimize crosslinkers: Test DSS vs. DTME for stabilizing transient protein interactions .

  • Use sequential elution: Stepwise pH elution (pH 2.5–8.0) to isolate tightly bound complexes .

  • Quantify recovery via SILAC-labeled internal standards for MS-based proteomics .

• How to design CRISPR mutants to study At4g08875 functional redundancy with paralogs?

  • Target conserved domains (e.g., Table 1) while preserving unique regions:

    Gene	Conserved Domain (AA)	Unique Region (AA)
    At4g08875	50-120 (Kinase-like)	150-200 (C-terminal loop)
    At3g45093	45-115 (Kinase-like)	180-220 (α-helix)

  • Perform phenotypic complementation with domain-swap constructs to assess functional overlap .

• How to analyze contradictory ChIP-seq and Y1H data for At4g08875-DNA interactions?

  • Evaluate antibody cross-reactivity: Test binding to truncated At4g08875 variants in vitro .

  • Apply competitive EMSA: Mix labeled DNA probes with increasing concentrations of unlabeled competitor DNA .

  • Use Bayesian statistical modeling to reconcile false-positive rates between techniques .

Critical Data Validation

• Interpreting non-linear Western blot bands for At4g08875:

  • Post-translational modifications: Treat extracts with λ-phosphatase or PNGase F to test phosphorylation/glycosylation .

  • Proteolytic degradation: Include protease inhibitors (e.g., PMSF + Complete™ EDTA-free) and compare fresh/frozen samples .

Methodological Pitfalls & Solutions

• Artifacts in co-immunoprecipitation experiments:

  • Problem: False-positive interactions due to antibody aggregation.

  • Solution: Pre-clear lysates with Protein A/G beads and include IgG isotype controls .

  • Validation: Reciprocal IP with tagged constructs (e.g., FLAG-At4g08875 vs. HA-interactor) .

• Quantifying At4g08875 expression under abiotic stress:

  • Normalize to housekeeping proteins (e.g., Actin) and total protein (via Coomassie stain) to control for loading errors .

  • Use RNA-seq correlation: Compare antibody signal with transcript levels (e.g., eFP Browser data) .

Epitope Characterization

• Mapping conformational vs. linear epitopes of At4g08875 Antibody:

  • Linear epitopes: Test antibody binding to overlapping 15-mer peptides (offset by 3 AA) in dot-blot assays .

  • Conformational epitopes: Compare native vs. denatured protein reactivity via ELISA .

Key Citations

• UniProt entry Q2V3K2 defines At4g08875 domains .

• Cross-reactivity controls adapted from anti-α-synuclein/Aβ antibody validation protocols .

• CRISPR design informed by Arabidopsis SYP32/SYP31 knockout studies .