FTSH3 Antibody

Here’s a structured FAQ collection for researchers working with FTSH3 antibodies, based on experimental methodologies and findings from academic studies:

Advanced Research Questions

How to resolve contradictions in FTSH3-substrate interaction data across studies?

• Case example: Discrepancies in FTSH3-PSST interaction assays (Y2H vs. Co-IP).

  • Solution: Combine orthogonal methods (e.g., BiFC for in vivo interaction vs. Y2H for direct binding) .

  • Table: Interaction assay outcomes

    Method	FTSH3-PSST Interaction	Mutation Impact (P415L/S70F)
    Co-IP	Positive	Interaction abolished
    Y2H	Positive	Interaction abolished
    BiFC	Positive in vivo	Reduced fluorescence signal

What strategies optimize FTSH3 antibody use in pulse-chase protein turnover assays?

• Protocol refinement:

  • Treat samples with cycloheximide to inhibit translation, then track PSST degradation over time .

  • Use rmb2 ciaf1 mutants (PSST S70F) to assess FTSH3 dependency .

• Critical controls:

  • Include ATPase-deficient FTSH3 mutants to confirm turnover mechanism.

How to integrate FTSH3 antibody data with structural studies of Complex I?

• Workflow:

  • Immunoprecipitate FTSH3-bound CI subunits using the antibody .

  • Perform cryo-EM on purified complexes to map disassembly sites.

  • Cross-reference with Alphafold predictions of PSST-FTSH3 binding interfaces .

Methodological Best Practices

Key validations for FTSH3 antibody in plant mitochondrial studies

• Specificity checks:

  • Test on fts3 knockout lines (no signal expected).

  • Compare with mitochondrial subfractionation (enrichment in membrane-bound fractions) .

• Quantitative thresholds:

  • ≥5-fold signal reduction in mutants vs. wild-type in Western blots.

Troubleshooting non-specific binding in FTSH3 immunohistochemistry

• Mitigation steps:

  • Pre-absorb antibodies with E. coli-expressed FTSH3 protein.

  • Use blocking buffers with 5% BSA + 0.1% Tween-20 to reduce background .

Data Interpretation Guidelines

Interpreting FTSH3 antibody results in oxidative stress conditions

• Key considerations:

  • FTSH3 preferentially degrades oxidized CI subunits .

  • Correlate antibody signals with ROS levels (e.g., H2O2 assays).

Reconciling FTSH3’s role in CI disassembly vs. prior reports on FTSH10

• Critical analysis:

  • FTSH3 forms homo-hexamers (not hetero-hexamers with FTSH10) during CI interaction .

  • Validate using FTSH10 antibodies in parallel Co-IPs .