Phos-tag SDS–PAGE and immunoblot analysis

Cell lysates were mixed with 4× SDS–PAGE sample buffer [250 mM Tris–HCl, pH 6.8, 8% by mass SDS, 40% by vol glycerol, 0.02% by mass Bromophenol Blue and 4% by vol 2-mercaptoethanol] and heated at 95°C for 5 min. Samples were supplemented with 10 mM MnCl₂ before boiling and then centrifuged for 1 min at 14 000 rpm. Phos-tag SDS–PAGE was carried out as described previously [42] with some modifications. Gels for Phos-tag SDS–PAGE consisted of a stacking gel [4% by mass acrylamide, 125 mM Tris–HCl, pH 6.8, 0.1% by mass SDS, 0.2% by vol N,N,N′,N′ tetramethylethylenediamine (TEMED) and 0.08% by mass ammonium persulfate (APS)] and a separating gel [8% by mass acrylamide, 375 mM Tris–HCl, pH 8.8, 0.1% (by mass SDS, 100 µM Phos-tag acrylamide, 200 µM MnCl₂, 0.1% by vol TEMED and 0.05% by mass APS]. Ten to forty microgram of samples were loaded and electrophoresed at 70 V for the stacking part (∼2 h) and then 120 V for 150 min with the running buffer [25 mM Tris–HCl, 192 mM glycine and 0.1% by mass SDS]. For immunoblot analysis, gels were washed for 10 min in the transfer buffer [48 mM Tris–HCl, 39 mM glycine and 20% by vol methanol] containing 10 mM EDTA and 0.05% my mass SDS three times, followed by one wash in the transfer buffer containing 0.05% SDS for 10 min. Proteins were electrophoretically transferred onto nitrocellulose membranes (Amersham Protran 0.45 µm NC; GE Healthcare) at 100 V for 180 min on ice in the transfer buffer without SDS/EDTA. Transferred membranes were blocked with 5% by mass non-fat dry milk dissolved in TBS-T [20 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.1% by vol Tween 20] at room temperature for 45 min. Membranes were then incubated with primary antibodies overnight at 4°C. After washing membranes in TBS-T, membranes were incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h. After washing membranes in TBS-T, protein bands were detected by exposing Amersham Hyperfilm to the membranes using an ECL solution Amersham ECL Western Blotting Detection Reagents (GE Healthcare).