Protean 2 xl 2 d cell apparatus

For IEF (isoelectricfocusing), 100 μg of each protein sample were loaded onto the strip gels, rehydrated for 12 h (18 cm, pH 4–7) with rehydration buffer (7 M urea, 2 M thiourea, 2% v/v CHAPS, 2% IPG buffer (pH 4–7). The protein samples were then electrofocused in a manifold cup-loading system with IPGphor (GE Healthcare, Piscataway, NJ, USA), and 2^nd dimension was carried out at 15 mA overnight using a PROTEAN II xl 2-D Cell apparatus (BIO-RAD, Hercules, CA, USA) following our previous described procedure52 (link).

Samples of the cellular protein extracts prepared as described above were subjected to two dimensional-polyacrylamide gel electrophoresis (2D-PAGE). One hundred μg of each protein sample was loaded onto the strip gels and rehydrated for 12 h (18 cm, pH 4–7) with rehydration buffer (7 M urea, 2 M thiourea, 2% v/v CHAPS, 2% IPG buffer, pH 4–7) (GE Healthcare, Piscataway, NJ, USA). The samples were then electrofocused in a manifold cup-loading system with IPGphor (GE Healthcare, Piscataway, NJ, USA) in the following sequential steps; pre-separation at 100 V for 1 h, 200 V for 1 h, 500 V for 1 h; application of the samples to the strip gels initially at 1,000 V for 4 h; gradient focusing from 1,000 V to 8,000 V for 30 min; and finally steady-state focusing at 8,000 V for 8 h. The separated strip gels were equilibrated with equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-Cl, pH 8.8, 30% glycerol) containing 65 mM DTT for 15 min. After a second equilibration with the same buffer containing 2.5% v/v iodoacetamide instead of DTT and a trace of bromphenol blue for 15 min; the equilibrated strip gels were applied to 1.0 mm thick 10% acrylamide gels and sealed with 0.25% (w/v) agarose. SDS-PAGE was carried out at 15 mA overnight using a PROTEAN IIxl 2-D Cell apparatus (BIO-RAD, Hercules, CA, USA).