Molecular imager gs 800tm calibrated densitometer

Cellular proteins were obtained by sonicating the bacteria (Ultrasonic Processor, Cole Parmer Corporation, USA) in the presence of protease inhibitors (10 mM PMSF, 1 mM EDTA; cycles: 1 min ON/1 min OFF) at 4°C. For 2D-PAGE, approximately 80 μg of protein for the analytical gels or 100–150 μg of protein for the preparative gels was solubilised, denatured, and reduced in sample buffer (4% CHAPS, 9 M urea, 70 mM l-dithiothreitol (DTT), 0.001% bromophenol blue, and 0.1% 3–10 ampholyte) and was used to rehydrate 11-cm, pH 4–7 IPG strips (ReadyStripTM, IPG strips, Bio-Rad, USA). IEF was carried out on a Multiphor II (Amersham Biosciences, UK) until 52,000 VH at 17°C. Prior to separation in the second dimension, IPG strips were equilibrated in a solution containing 6 M urea, 30% (v/v) glycerol, 50 mM Tris-base pH 8.8, and 2% (w/v) SDS. The strips were equilibrated first for 15 min with 70 mM DTT and then for 15 min with 120 mM iodoacetamide. The second-dimension electrophoresis was performed using a 12.5% polyacrylamide gel (Hoefer SE-600, Amersham Biosciences, UK) with a voltage gradient of 50–150 V for approximately four hours. Once fixed, the proteins were silver-stained and the gel images were then captured in a digital format for analysis (Molecular Imager GS-800TM Calibrated Densitometer, Bio-Rad, USA).