FTSH4 Antibody

Basic Research Questions

How to validate FTSH4 antibody specificity for plant mitochondrial studies?

• Perform Western blot analysis using mitochondrial isolates from wild-type and ftsh4 knockout mutants (e.g., Arabidopsis or Synechocystis). A specific antibody should show:

  • Strong signal in wild-type mitochondrial fractions

  • Absence of signal in ftsh4 mutants

• Include controls for cross-reactivity with other FTSH proteases (e.g., FTSH1-3) by comparing migration patterns on SDS-PAGE .

What experimental applications are optimal for FTSH4 antibodies?

• Localization studies: Use immunofluorescence or GFP-tagged FTSH4 strains to visualize suborganellar distribution (e.g., thylakoid periphery in chloroplasts or inner mitochondrial membrane clusters) .

• Co-immunoprecipitation (Co-IP): Identify interaction partners like Sll1106 in Synechocystis or HSP23.6 in Arabidopsis using mild detergents (e.g., digitonin) to preserve membrane protein complexes .

Advanced Research Questions

How to resolve contradictions in FTSH4 expression levels under stress conditions?

• Experimental design: Compare temporal dynamics (e.g., 0–72 hr post-stress) and tissue-specific responses. Example findings:

  Condition	FTSH4 Protein Level	Key Partner Proteins	Citation
  High light (24h)	Decreased	HliD ↑
  Oxidative stress	Increased	HSP23.6, MPC4 ↓

• Methodological adjustments: Use quantitative proteomics (iTRAQ) alongside immunoblotting to distinguish degradation vs. synthesis rates .

What strategies identify novel FTSH4 substrates in organello?

• Substrate trapping: Employ catalytically inactive FTSH4 mutants (e.g., FTSH4ᴱ⁴⁷⁶Q) to capture transiently bound proteins, followed by mass spectrometry (Table 1) :

  Substrate	Function	Degradation Kinetics in ftsh4
  HSP23.6	Chaperone	3x slower turnover
  MPC4	Pyruvate carrier	2.5x accumulation

• Validation: Conduct in organello degradation assays using isolated mitochondria from mutants vs. wild-type .

How to interpret FTSH4 antibody signals in mutant complementation lines?

• Case study: In Synechocystis F4CF overexpression strains:

  • Expect higher FTSH4 signal but lower HliD levels under high light due to protease-mediated regulation .

  • Monitor autoproteolysis products via 2D gel electrophoresis to distinguish full-length vs. degraded forms .

Methodological Considerations

Optimizing FTSH4 detection in membrane-rich samples

• Solubilization: Use 1–2% β-DM or LMNG for chloroplast/mitochondrial membranes to prevent aggregation .

• Crosslinking: Apply formaldehyde fixation before immunofluorescence to stabilize transient FTSH4 clusters in thylakoids .

Addressing false positives in Co-IP experiments

• Negative controls: Include pulldowns from untagged strains or IgG-only columns (e.g., WT Synechocystis membranes in ).

• Quantitative thresholds: Filter MS data using iBAQ scores (e.g., ≥4-fold enrichment over background) .

Emerging Research Frontiers

Does FTSH4 participate in organelle membrane remodeling?

• Key evidence:

  • FTSH4 co-purifies with cardiolipin synthase regulators in Arabidopsis .

  • ftsh4 mutants exhibit giant mitochondria with reduced fission, linked to defective mitophagy .

• Method recommendation: Combine lipidomics with super-resolution microscopy to map FTSH4’s spatial relationship with membrane curvature sensors.

Can FTSH4 antibody distinguish between assembly vs. repair roles in PSII biogenesis?

• Experimental approach:

  • Pulse-chase labeling with ³⁵S-methionine in Synechocystis.

  • Immunoprecipitate early PSII assembly intermediates (e.g., D1/D2 complexes) using FTSH4 antibody .

  • Compare turnover rates in Δftsh4 vs. WT under photo inhibitory conditions.