At2g29830 Antibody

Basic Research Questions

Q1: How can researchers validate the specificity of an At2g29830 antibody in Arabidopsis thaliana protein extracts?

• Methodological Answer:

  • Western Blot: Use knockout mutants (e.g., T-DNA insertion lines) to confirm absence of signal. Include tissue-specific extracts (root, leaf, flower) to assess isoform expression.

  • Immunoprecipitation (IP): Couple with mass spectrometry to identify co-purified proteins (see for FLC chromatin interaction workflows).

  • ELISA: Test recombinant At2g29830 protein against non-target F-box family proteins (e.g., SKP1-like proteins) to rule out cross-reactivity (methods adapted from ).

Q2: What experimental controls are critical when studying At2g29830’s role in ubiquitination pathways?

• Key Controls:

  • Negative Controls: Use at2g29830 knockout lines or RNAi-silenced plants.

  • Positive Controls: Include known F-box protein interactors (e.g., ASK1/ASK2) in co-IP assays.

  • Buffer Specificity: Pre-clear lysates with protein A/G beads to reduce non-specific binding (as in ).

Advanced Research Questions

Q3: How can researchers resolve contradictory data on At2g29830’s substrate specificity across studies?

• Analytical Framework:

  • Orthogonal Assays: Compare yeast-two-hybrid, in vitro pull-down, and in vivo BiFC results ( used similar strategies for JMJ30/JMJ32).

  • Post-Translational Modifications (PTMs): Screen for phosphorylation or ubiquitination sites via mutagenesis (e.g., alanine scanning).

  • Context Dependency: Test substrate interactions under stress conditions (heat, drought) to identify environment-specific roles.

Q4: What computational tools are recommended for predicting At2g29830’s interactome?

• Tools & Workflows:

  • Phylogenetic Profiling: Use PLAZA 5.0 or Phytozome to identify co-evolving partners.

  • Molecular Dynamics (MD) Simulations: Model protein-ligand interactions using SWISS-MODEL or AlphaFold (methods in ).

  • Co-Expression Networks: Leverage ATTED-II or AraNet to prioritize candidates for experimental validation.

Methodological Challenges & Solutions

Q5: How to address non-specific binding of At2g29830 antibody in immunofluorescence assays?

• Troubleshooting Steps:

  • Pre-Absorption: Incubate antibody with E. coli-expressed At2g29830 protein (see for recombinant protocols).

  • Blocking Optimization: Use 5% BSA + 0.1% Tween-20 in PBS, supplemented with plant-specific protease inhibitors.

  • Dual Labeling: Combine with GFP-tagged At2g29830 lines (e.g., pAt2g29830::At2g29830-GFP) to confirm colocalization.

Data Integration & Interpretation

Q6: How to reconcile transcriptomic and proteomic data discrepancies for At2g29830?

• Integrated Approach:

  • Multi-Omics Correlation: Map RNA-seq, ribo-seq, and proteomics data using platforms like OmicsBox.

  • Half-Life Analysis: Treat plants with cycloheximide to measure protein turnover rates (as in for JMJ30 stability assays).

  • Subcellular Fractionation: Isolate nuclei/cytoplasm to detect compartment-specific protein accumulation.

Cross-Disciplinary Applications

Q7: Can At2g29830 antibody be repurposed for studying non-plant systems?

• Validation Strategy:

  • Phylogenetic Alignment: Check for conserved epitopes in orthologs (e.g., human F-box proteins like SKP2).

  • Functional Complementation: Express At2g29830 in yeast fbxΔ mutants to test rescue of ubiquitination defects.

Key Data Table: At2g29830 Antibody Validation Metrics