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Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A

ent stability. The reagents for casting twelve 1.0 mm, 916% gels were as follows: for the 9% solution, 114 ml of 30% acrylamide (37.5:1 acrylamide:bis-acrylamide, Roche Diagnostics), 142.2 ml 1 M Tris, pH 8.8, 120 ml water, 1.91 ml 20% SDS, 1.26 ml 10% ammonium persulfate, and 252 l TEMED; for the 16% solution, 197 ml acrylamide, 138 ml Tris, 32 ml 50% glycerol, 1.85 ml SDS, 1.23 ml ammonium persulfate, and 245 l of TEMED. TEMED was added to the solutions at the last moment and the gradients pumped into the bottom of the casting chamber over about 6 min. Each gel was immediately overlaid with 0.75 ml of water-saturated butanol, and once set, transferred to 4C for storage overnight before use.

Equilibration and Loading of IPG Strips
IPG strips were equilibrated in 10 ml of 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and Bromophenol Blue (trace), first for 15 min with 100 mg dithioerythritol (DTE), and then for 15 min in a second 10 ml of equilibration buffer with 250 mg iodoacetamide. Strips were then placed on the top surface of the SDS-PAGE gels and sealed with agarose in SDS-PAGE running buffer (Berkelman and Stenstedt 1998).

Running Conditions
The loaded gels (11) were placed into the Dodeca chamber with 23 L of running buffer (made by dilution of Bio-Rad Tris/glycine/SDS buffer, catalog #161-0772) precooled to 18C using a Techne FC-200 flow cooler and circulator. (Another gel was run separately.) Voltage was applied at 50 V for 0.5 hr, then at 200 V until the Bromophenol Blue line reached the bottom of the gels (about 6 hr). The Dodeca cell is straightforward to use when the PowerPac power supply is programmed at constant voltage with no current limit. Unde
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