Alkaline phosphatase conjugated anti rabbit igg

Proteins were separated by 12.5% or 15% SDS-PAGE on a 0.1% SDS-Tris-glycine running buffer system and stained with Coomassie blue or silver using a silver staining kit (Daiichi Pure Chemical, Tokyo, Japan). For western blotting, SDS-PAGE gels were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) in a buffer containing 20% methanol, 25 mM Tris (pH 8.3), and 192 mM glycine. Membranes were incubated for 30 min in blocking buffer consisting of 1% Western Blocking Reagent (Roche Molecular Diagnostics, Indianapolis, IN, USA) in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, adjusted to pH 7.5 with NaOH). The membranes were incubated with rabbit antiserum (1:1000 in maleic acid buffer), and then alkaline phosphatase-conjugated anti-rabbit IgG (Bio-Rad) diluted 1:10,000 in maleic acid buffer. Bands were visualized using a chemiluminescent substrate kit (Bio-Rad).

GC cells were lysed with Pierce RIPA buffer (Thermo Scientific; #89900). Proteins were separated on SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes, followed by incubation with antibodies dissolved in Tris-buffered saline (TBS) containing 2% skim milk and 10% Tween 20. The primary antibodies used in this study were mouse monoclonal anti-Twist1 (1:100, Santa Cruz Bio-technology; #sc-81417), rabbit polyclonal anti-Sp1 (1:200, Active Motif; #39058), and mouse monoclonal anti-α-tubulin (1:200, Santa Cruz; #sc-8035) antibodies. The secondary antibodies were alkaline phosphatase-conjugated anti-rabbit IgG (1:3000, Bio-Rad Laboratories; #1706518) and anti-mouse IgG (1:3000, Bio-Rad Laboratories; #1706520). Blots were developed with ImmunoStar AP Substrate (Bio-Rad Laboratories; #1705018).