24cm immobiline drystrips

Protein separation of three biological replicates per strain was performed using 600μg of protein. This amount of protein was diluted in DIGE buffer (as described above and supplemented with IPG buffer pH 3–10 NL (0.8%, v/v) and 60mM DTT) to a final volume of 450μl. IEF was then performed using the IPGphor system (using cup loading and manifold) and 24cm immobiline drystrips with a non-linear pH gradient from 3 to 10 (all from GE Healthcare, Sweden). The protocol consisted of a sequence of 7 steps as follows: rehydration of the strips (50μA/strip at 20°C) was carried out for 12 h at 30V, followed by a step-and-hold running condition at 100Vh (3h), step from 300V to 600Vhr, step from 500V to 500Vhr, gradient at 3500V (2h), step from 3500V to 7000Vhr, gradient at 10000Vh (3h), and a final step at 10000Vh (5h), with a total of 77,7kVh.

For analytical 2D-DIGE analysis 50 μg each of Cy3-, Cy5-, and Cy2-labeled protein samples were mixed together (total 150 μg of protein). The DIGE sample buffer [7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, and 0.5% IPG buffer (GE Healthcare)] was added to bring the volume to 450 μl, and the samples were then applied to 24-cm Immobiline Drystrips (GE Healthcare) and rehydrated overnight. IEF was carried out on an Ettan IPGphor II (GE Healthcare) at 20⋅C with a maximum of 50 μA/strip and the following setting: 500 and 1000 V each for 1 h, a gradient increase to 8000 V over 3 h, and remaining at 8000 V until an accumulated voltage of reaching the desired total V-h (72,000 for pH 4–7 IPG strips). After IEF, IPG strips were equilibrated in equilibration buffer [6 M urea, 30% (w/v) glycerol, 2% SDS, 50 mM Tris-HCl, pH 8.0] first with 0.5% DTT and then with 2% iodoacetamide each for 15 min. The equilibrated strips were then transferred to 12.5% SDS-PAGE gels for the second dimension electrophoresis using the Ettan Dalt-six (GE Healthcare/Amersham Biosciences) vertical unit. SDS-PAGE was run overnight: the first step at 80 V, for 1 h, the second step at 120 V, until the bromophenol blue dye front reached the bottom of gel. Four biological trials were performed at each time point.