At2g04230 Antibody

FAQs for Researchers on At2g04230 Antibody in Academic Research

How can I validate the specificity of At2g04230 antibodies in plant protein studies?

Methodological Answer:

• Knockout (KO) Controls: Use Arabidopsis lines lacking the At2g04230 gene (e.g., T-DNA insertion mutants) to confirm absence of signal in Western blot (WB) or immunofluorescence (IF) .

• Orthogonal Validation: Combine WB with targeted proteomics or mRNA expression data (e.g., RT-qPCR) to correlate protein and transcript levels .

• Cross-Reactivity Testing: Screen against related F-box proteins (e.g., D5BF2/At3g23970) using overexpression lines .

Example Workflow:

• Perform WB on wild-type and KO plant lysates.

• Validate with IF on tissues with known expression (e.g., root tips for D5BF1).

• Compare results to public proteomic databases (e.g., TAIR, Araport).

What are the recommended controls for immunolocalization of At2g04230 in plant tissues?

Methodological Answer:

• Negative Controls:

  • Tissues/cell lines lacking At2g04230 (e.g., d5bf1 mutants) .

  • Pre-immune serum or isotype-matched irrelevant antibodies .

• Positive Controls:

  • Transgenic lines overexpressing GFP/D5BF1 fusions .

  • Tissues with high endogenous expression (e.g., vascular bundles) .

Pitfalls to Avoid:

• Non-specific binding to epithelia or cell walls (common in plant IF) .

• Overfixation, which masks epitopes .

How should At2g04230 antibody titrations be optimized for flow cytometry?

Methodological Answer:

• Titration Curve: Test antibody dilutions (e.g., 1:50–1:1000) on cells with high/low target expression .

• Validation Metrics:

  • Signal-to-noise ratio ≥ 3:1.

  • Minimal overlap between positive and negative populations .

• Reprodubility: Use the same batch of antibodies and fixation protocols across experiments .

Example Data:

How do I resolve contradictions in At2g04230 antibody performance across studies?

Methodological Answer:

• Antigen Specificity: Confirm the immunogen sequence aligns with At2g04230’s C-terminal domain (critical for F-box function) .

• Assay-Specific Validation: Antibodies may work in WB but fail in IP/IF due to conformational epitopes .

• Literature Audit: Cross-reference studies using the same clone (e.g., YCharOS data showing 50–75% failure rates for commercial antibodies) .

Case Study:
A 2023 study found that 73 antibodies targeting human proteins were discontinued due to nonspecificity, highlighting the need for third-party validation .

What experimental designs are optimal for studying At2g04230 in plant-microbe interactions?

Methodological Answer:

• Pathogen Challenge Models: Use Agrobacterium tumefaciens infection assays to study D5BF1-mediated VirD5 degradation .

• Functional Redundancy: Test double mutants (e.g., d5bf1 d5bf2) to bypass genetic compensation .

• Dynamic Localization: Combine IF with subcellular fractionation to track D5BF1 during infection .

Key Findings:

• Agrobacterium suppresses D5BF1 expression via unknown effectors, enhancing virulence .

• D5BF1 degrades VirD5 via the 26S proteasome, reducing tumorigenesis by 40–60% .

How can I integrate At2g04230 antibody data into multi-omics workflows?

Methodological Answer:

• Proteogenomic Alignment: Cross-validate antibody data with RNA-seq (e.g., eFP Browser) and phosphoproteomics .

• Machine Learning: Train models to predict antibody performance using features like epitope accessibility and homology .

Workflow Table:

Key Recommendations:

• Prioritize recombinant antibodies, which show higher specificity in head-to-head studies .

• Use CRISPR-edited controls for critical validations .

• Publish full validation data (e.g., uncropped blots, IF parameters) to enhance reproducibility .