At3g49150 Antibody

Here’s a structured collection of FAQs tailored for academic researchers working with the At3g49150 antibody, incorporating methodological guidance and evidence-based insights:

What experimental applications is the At3g49150 antibody validated for?

The At3g49150 antibody (Uniprot: Q9M3B7) is validated for ELISA and Western blot (WB) applications in Arabidopsis thaliana systems . For WB, ensure lysates are prepared under denaturing conditions to expose the recombinant epitope. Include positive controls (e.g., transgenic lines overexpressing At3g49150) and negative controls (knockout mutants) to confirm specificity .

How should storage conditions be optimized to preserve antibody functionality?

• Short-term: Store at -20°C in 50% glycerol to prevent freezing .

• Long-term: Aliquot and store at -80°C; avoid repeated freeze-thaw cycles to prevent aggregation .

• Post-reconstitution: Dilute in PBS (pH 7.4) with 0.03% Proclin 300 to inhibit microbial growth .

What validation steps ensure antibody specificity in plant proteomics?

• Epitope mapping: Perform competitive ELISA with recombinant At3g49150 protein to confirm binding .

• Cross-reactivity checks: Test against lysates from phylogenetically related species (e.g., Brassica napus) to rule out nonspecific binding .

• Knockout validation: Compare signal intensity in wild-type vs. at3g49150 T-DNA insertion mutants .

How can researchers resolve contradictory results in Western blotting using this antibody?

For quantitative discrepancies, integrate mass spectrometry to verify protein identity .

What strategies enable epitope characterization for structural studies?

• HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry): Identify regions protected by antibody binding .

• Computational docking: Use tools like HADDOCK to predict antibody-antigen interfaces (e.g., hydrophobic patches near the active site, as seen in PR3-ANCA studies) .

• Peptide arrays: Synthesize overlapping 15-mer peptides spanning At3g49150 to map linear epitopes .

How can this antibody be integrated into systems biology workflows?

• Co-immunoprecipitation (Co-IP): Crosslink with DSS to stabilize transient interactions; validate partners via SILAC-based proteomics .

• Spatial localization: Combine with GFP-tagged constructs and confocal microscopy to resolve subcellular distribution .

• Multi-omics correlation: Pair WB data with RNA-seq profiles to dissect post-transcriptional regulation .

Data Contradiction Analysis Framework

When reconciling conflicting results (e.g., absence of signal in mutant rescue lines):

• Technical variables:

  • Compare antibody lot-to-lot variability using internal reference samples .

  • Validate secondary antibody compatibility (e.g., anti-rabbit IgG cross-reactivity) .

• Biological context:

  • Assess tissue-specific isoform expression via RNA-Seq (e.g., alternative splicing in roots vs. leaves) .

  • Investigate post-translational modifications (e.g., phosphorylation) that may obscure epitopes .