For contamination-free apoplast isolation from 4–5-week-old Arabidopsis thaliana grown under 12h day/12h night, consider the following optimized protocol:

1. Vacuum Infiltration Parameters

• Buffer Choice: Use ice-cold MES buffer (e.g., 10 mM MES-KOH, pH 6.0) with 100 mM KCl and 0.2 mM CaCl₂ to maintain ion balance.
• Vacuum Pressure: Apply −60 to −80 kPa (0.6–0.8 bar or ~450–600 mmHg) for 3–5 minutes in a desiccator to allow buffer infiltration into the apoplast. Avoid over-infiltration to prevent tissue rupture.
• Release Phase: Slowly release the vacuum over 30–60 seconds to prevent tissue collapse.

2. Centrifugation Parameters for Apoplast Extraction

• Centrifuge Conditions:Use 0.22 μm filter-sterilized buffer to avoid microbial contamination. 500–1000 × g for 10–15 minutes at 4°C in a swinging-bucket rotor. Avoid exceeding 1200 × g, as higher forces may rupture cells, leading to cytoplasmic contamination.
• Filtration Step (Optional): To ensure purity, filter the collected apoplastic wash through a 0.45 μm filter.

3. Purity Control and Contamination Checks

• Chloroplastic Contamination: Check for Rubisco (RbcL or RbcS) via Western blot or qPCR.
• Cytoplasmic Contamination: Use cytoplasmic markers like GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or UDPG pyrophosphorylase via enzyme activity assays or Western blot.
• Apoplast-Specific Markers:α-Mannosidase (apoplastic enzyme) activity assay. PEROXIDASE activity as apoplast-specific control.

4. Additional Tips

• Leaf Handling: Avoid over-handling leaves to minimize wound-induced contamination.
• Pre-Cleaning: Rinse leaves in sterile distilled water before infiltration.
• Use Controls: Compare isolated apoplastic fluid with cytoplasmic/extracellular markers from total leaf lysates.

To achieve contamination-free apoplast isolation from 4-5-week-old Arabidopsis plants, follow these optimized protocols and parameters based on established methods:

Vacuum Infiltration Parameters

Centrifugation Protocol

Purity Validation

Additional Tips

• Pre-infiltration: Ensure plants are well-watered 24 hours before isolation to maximize apoplastic fluid yield
• Infiltration buffer: Use ice-cold, isotonic solutions (e.g., 10 mM MES-KOH pH 6.0) to stabilize proteins and metabolites
• Post-centrifugation: Process samples immediately or store at 4°C for ≤48 hours to prevent degradation.