Protean plus dodeca cell

After IEF, the IPG strips were either stored at −80°C or transferred to 10 mL of equilibration buffer (6 M urea, 30% w/v glycerin, 2% w/v SDS, 50 mM Tris–HCl pH 8.8) with 2% w/v DTT and 0.5% v/v bromophenol blue solution (0.25% w/v bromophenol blue, 1.5 M Tris–HCl pH 8.8, 0.4% w/v SDS) and incubated for 20 min at room temperature. Strips were removed and incubated in equilibration buffer with 4% w/v iodoacetamide and 0.5% v/v bromophenol blue solution for further 20 min at room temperature. Finally, the strips and 10 μL SDS-PAGE molecular weight standard on filter paper were placed on top of the 20 cm x 20.5 cm 12% second-dimension gel (12% v/v acrylamide/bis solution, 375 mM Tris, pH 8.8, 0.1% v/v SDS, 1/2000 TEMED, 0.05% v/v APS). Both were fixed in place with a 0.5% w/v agarose overlay. Gels were run in PROTEAN Plus Dodeca cell (Bio-Rad) at 70 V for approximately 14 h, followed by 200 V until the bromophenol blue dye reached the bottom of the gel. The running buffer (25 mM Tris, 0.2 M glycin, 0.1% SDS) was cooled externally to 16°C.
Gels/proteins were fixed overnight in 30% ethanol, 2% phosphoric acid, and washed 3 x 20 min with 2% phosphoric acid. The gels were equilibrated with 15% ammoniumsulfate, 18% ethanol, 2% phosphoric acid for 15 min and finally stained with colloidal Coomassie Blue for 48 h.

The IPG strips were prepared and equilibrated for the 12.5% (wt/vol) polyacrylamide gels (200 mm x 200 mm x 1 mm) as described previously [23 (link)]. To this end, strips were fixed onto the gels with sealing solution (1% agarose, wt/vol, spatula-tip of bromophenol blue) and the SDS-polyacrylamide gel electrophoresis (PAGE) was performed in a DODECA Cell (PROTEAN^® plus DODECA^™ Cell, Biorad, Hercules, USA) filled with buffer (25 mM Tris/HCl, 192 mM glycine, 0.1% (wt/vol) SDS). PAGE was carried out at 15°C, and 5 V per gel were applied for 1.5 h. Afterwards, 20 V per gel were used until the dye front reached the end of the gels. The gels were stained for approx. 16 h with staining solution containing 0.4% Serva Blue G 250 (wt/vol), 45% methanol, and 9% acetic acid. Destaining was performed with a solution composed of 33% (vol/vol) methanol and 10% (vol/vol) acetic acid for 10 h and afterwards, the gels were stored in 10% acetic acid.

After preparation of the IPG strips, 12.5% (wt/vol) polyacrylamide gels (200 mm x 200 mm x 1 mm) were produced as described before [22 (link)]. IPG-strips were equilibrated for 10 min in SDS-equilibration buffer (1.5 M Tris/HCl pH 8.8, 7M urea, 2% SDS, 34.5% glycerol, and a pinch of bromophenol blue) and then for another 10 min in SDS-equilibration buffer supplemented by iodoacetamide. The strips were washed in electrode buffer (25 mM Tris/HCl, 192 mM glycine, 0.1% (wt/vol) SDS), which was also used for 2D-PAGE in a DODECA Cell (PROTEAN plus DODECA Cell, Biorad, Hercules, USA), and fixed onto the gels with sealing solution (1% agarose (wt/vol) in electrode buffer and a pinch of bromophenol blue). The PAGE was carried out with 5 V per gel for one hour and afterward with 20 V per gel until the run was finished. Afterward, gels were stained overnight (0.4% Serva Blue G 250 (wt/vol), 45% methanol (vol/vol), and 9% acetic acid (vol/vol)) and destained for 10 h (33% (vol/vol) methanol and 10% (vol/vol) acetic acid). Gels were stored in 10% acetic acid (vol/vol).