At4g27190 Antibody

Basic Research Questions

What experimental approaches validate At4g27190 antibody specificity in plant immunity studies?

• Methodological steps:

  • Perform western blotting using Arabidopsis wild-type and At4g27190 knockout mutants to confirm absence of signal in mutants .

  • Use immunolocalization with confocal microscopy to verify subcellular localization patterns matching the protein’s predicted nuclear/cytoplasmic distribution .

  • Validate via peptide competition assays by pre-incubating antibodies with the At4g27190-derived antigen (e.g., residues 200-250 of the NB-ARC domain) .

How is At4g27190 antibody used to study disease resistance mechanisms?

• Experimental design:

  • Combine co-immunoprecipitation (Co-IP) with mass spectrometry to identify interacting partners (e.g., signaling kinases) in pathogen-challenged Arabidopsis .

  • Quantify protein expression kinetics using ELISA during Pseudomonas syringae infection time courses .

  • Correlate antibody-detected protein levels with transcript data (RT-qPCR) to assess post-transcriptional regulation .

Advanced Research Challenges

How to resolve contradictions between antibody detection and phenotypic data in At4g27190 mutants?

• Troubleshooting framework:

  Issue	Solution	Supporting Data Source
  Unexpected bands in western blots	Test cross-reactivity against homologous NB-ARC proteins (e.g., At4g33300) using recombinant antigens	: 34 Arabidopsis antibodies listed with cross-reactivity risks
  Weak signal in immunofluorescence	Optimize fixation protocols (e.g., paraformaldehyde concentration) to preserve conformational epitopes	: Arabinanase-sensitive epitope validation example
  Discrepancy in pathogen response	Perform allele-specific complementation assays with At4g27190 variants to confirm antibody-target interaction	: Gene-specific functional annotation

What integrated methods link At4g27190 antibody data to systemic acquired resistance (SAR)?

• Multi-omics strategy:

  • Map antibody-based protein localization changes during SAR using tissue-specific fractionation (nuclear vs. cytoplasmic) .

  • Pair with phosphoproteomics to identify post-translational modifications (e.g., phosphorylation at Ser-158) affecting activity .

  • Validate findings via electrophoretic mobility shift assay (EMSA) to test DNA-binding capacity in immune-activated states .

Technical Optimization

How to adapt protocols for At4g27190 antibody in low-abundance scenarios?

• Signal amplification:

  • Use tyramide-based amplification in immunohistochemistry (e.g., 10x signal enhancement in vascular tissues) .

  • Optimize chemiluminescent substrates (e.g., SuperSignal™ West Femto) for western blotting of root extracts .

  • Validate with fluorescence correlation spectroscopy (FCS) to quantify detection limits .

What controls are critical for ChIP-seq using At4g27190 antibody?

• Essential controls:

  • IgG isotype control: Prevents false positives from nonspecific binding .

  • Input DNA normalization: Accounts for chromatin accessibility biases .

  • Knockout validation: Compare peaks in wild-type vs. At4g27190 mutants to confirm target specificity .

Data Interpretation

How to reconcile conflicting transcript-protein expression ratios in stress conditions?

• Analytical pipeline:

  • Apply Spearman’s rank correlation to transcriptome-proteome datasets (>20 biological replicates) .

  • Use cycloheximide chase experiments to measure protein half-life under oxidative stress .

  • Model regulatory networks with Bayesian inference to identify upstream miRNAs or ubiquitin ligases .

What statistical frameworks address variability in antibody-based quantitation?

• Robust analysis:

  • Employ mixed-effects models to account for batch effects in large-scale screens .

  • Use Manders’ overlap coefficient for colocalization studies in confocal datasets .

  • Apply Grubbs’ test to remove outliers in ELISA absorbance readings .