Processor plus

Isoelectrofocusing (IEF) was performed using a 13 cm IPG strips (GE Healthcare) with a linear gradient ranging from 3 to 10: 200 μg of proteins was loaded by in gel rehydration. IEF was performed using IPGphor (GE Healthcare) at 20 °C, with 83,000 Vhrs. After IEF, the strips were incubated at room temperature in 6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris-HCl, pH 8.6, supplemented at first with 2% w/v DTT for 15 min and subsequently with 4.5% w/v iodoacetamide for 15 min. They were then loaded at the top of 1.0 mm vertical second dimension gels. SDS-PAGE was performed on 11.5% T and 3.3% C acrylamide (Biorad Acrylamide) homogeneous gels. The running buffer was 2.5 mM Tris, 192 mM glycine, 0.1% SDS. The running conditions were 11°C, 600 V constant voltage, 20 mA/gel, 60 W for 15 min and 11°C, 600 V constant voltage, 40 mA/gel, 80 W for about 2.5 h. Molecular weight markers were from the Low Mr Electrophoresis Calibration Kit (GE Healthcare). The gels were automatically stained using Processor Plus (Amersham Biosciences) with freshly prepared Neuhoff stain (Colloidal Coomassie Blue) ( Neuhoff, Arold, & Ehrhardt, 1988). They were digitized with the Personal Densitometer SI (Amersham Biosciences) and then stored after dehydration in a GD 2000 Vacuum Gel Dryer System (GE Healthcare).

Isoelectrofocusing (IEF) was performed using 13 cm IPG strips (GE Healthcare) with a linear gradient ranging from 3 to 10: 200 µg of proteins were loaded by in gel rehydration method. IEF was performed by using IPGphor (GE Healthcare, USA) at 20°C, with 83000 Vhrs. After IEF, the strips were incubated at room temperature in 6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris-HCl, pH 8.6, supplemented at first with 2% w/v DTT for 15 minutes and subsequently with 4.5% w/v iodoacetamide for 15 min. They were then loaded at the top of 1.0 mm vertical second dimension gels. SDS-PAGE was performed on 11.5% T and 3.3% C acrylamide (Biorad Acrylamide, USA) homogeneous gels. The running buffer was 2.5 mM Tris, 192 mM glycine, 0.1% SDS. The running conditions were 11°C, 600V constant voltage, 20 mA/gel, 60W for 15 min and 11°C, 600V constant voltage, 40 mA/gel, 80W for about 2.5 h. Molecular weight markers were from the Low Mr Electrophoresis Calibration Kit (GE Healthcare, USA). The gels were automatically stained using the Processor Plus (Amersham Biosciences, USA) with freshly prepared Neuhoff stain (Colloidal Coomassie Blue) (Neuhoff et al., 1988) .
They were digitized with the Personal Densitometer SI (Amersham Biosciences, USA) and then stored after dehydration in a GD 2000 Vacuum Gel Dryer System (GE Healthcare, USA).