Zn2+ Phos-tag SDS-PAGE

Whole cell lysates were prepared with RIPA buffer as described previously (20,21). Samples were mixed with a half volume of SDS-PAGE sample buffer [195 mM Tris-HCl (pH. 6.8), 30% glycerol, 15% 2-mercaptoethanol, 3% SDS, and 0.10% bromophenol blue], and then heated at 95°C for 5 min. The acrylamide pendant Phos-tag ligand and two equivalents of ZnCl₂ were added to the separating gel before polymerization. The running buffer for Zn²⁺ Phos-tag electrophoresis consisted of 100 mM Tris and 100 mM MOPS containing 0.1% SDS and 5.0 mM sodium bisulfite. Furthermore, the gel was washed in solution containing 25 mM Tris, 192 mM glycine, 10% methanol, and 1.0 mM EDTA for 20 min, then washed using a solution containing 25 mM Tris, 192 mM glycine, and 10% methanol for 20 min.