Figure 1.

Evaluation of vacuolar membrane purity by western blot analysis (A) and fractionation strategy for cauliflower tonoplast proteins (B). A, Purified vacuoles were treated with five different extraction methods: KI and alkaline (NaOH) washing, acetone and C/M extraction, and treatment with sodium phosphate pH 6 followed by C/M pH 6 extraction, subjected to one-dimensional SDS-PAGE followed by in gel digestion and LC/MS/MS. B, For analysis of purification 10 μg of total membrane proteins (totM) and tonoplast proteins (vacM) were loaded on each lane. Compartment specific antibodies were used for western-blot analysis: chloroplastic ATPase α-subunit (ATP-α; 55 kD), luminal binding protein (BIP; 73 kD), plasma membrane aquaporin (PAQ1; 30 kD), and γ-TIP (25 kD).