Ettan ipgphor 2 isoelectric focusing system

Cells were lysed in IEF lysis buffer (8 M urea, 1% NP-40, 10 mM DTT) with fresh protease and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated by isoelectric focusing using the Ettan IPGphor II isoelectric focusing system (GE) and separated by molecular weight using 4–12% Bis-Tris ZOOM NuPAGE gel (Invitrogen). A 7 cm Immobiline DryStrip (pH 3–10) was rehydrated in 0.5% IPG DeStreak rehydration buffer for 24 h (GE). Two hundred micrograms of whole cell lysate were mixed with DeStreak buffer and loaded on a Immobiline DryStrip, and IEF was performed according to manufacturer’s instructions. Following IEF, the strip was equilibrated in 1X LDS loading buffer with 1X DTT (Invitrogen) and loaded into the single well of the ZOOM NuPAGE gel and the standard Western protocol was followed.

The bacteria were treated with ursolic acid (Sigma, St. Louis, MO, USA) at a concentration corresponding to 4 × MIC for 60 min according to previous reports [9 (link),16 (link)]. This concentration decreases the growth rate of a mid-log-phase culture by 50% (Table 3) and could therefore be expected to trigger major changes in protein expression. Two-dimensional gel electrophoresis was performed by using the Ettan™ IPGphor II Isoelectric Focusing system (GE Healthcare, Piscataway, NJ, USA) according to the instructions of the manufacturer. Protein samples (300 μg) were separated by using IPG strips in pH range of 3–10. Isoelectric focusing was performed by using 7 M urea, 2 M thiourea, 4% CHAPS and 40 mM DTT with increasing voltage. Rod gels were soaked for 15 min at ambient temperature in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 10 mg/mL DTT) and applied to a second dimension using a commercially available 12% Tris-Glycine SDS gel (13 cm × 13 cm × 1.0 mm) (Pharmacia, Uppsala, Sweden) with an Ettan™ DALTsix Large Vertical System (GE Healthcare, Uppsala, Sweden). Image was scanned by ImageScanner™ (GE Healthcare, Uppsala, Sweden) and analyzed by ImageMaster™ 2D Platinum (GE Healthcare, Uppsala, Sweden).