Rabbit bax antibody

Bax and Bcl-2 expressions were detected by western blot analysis, as described previously (Shin et al., 2016 (link)). The tissues were homogenized with lysis buffer with 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl·6H₂O, 1 mM EGTA, 1 mM PMSF, 1 mM Na₂VO₄, and 100 mM NaF, and then centrifuged at 14,000 rpm during 30 min. Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to measure protein content. Protein with 30 μg was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. As primary antibodies, mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology), and rabbit Bax antibody (1:1,000; Santa Cruz Biotechnology) were used. Horseradish peroxidase-conjugated anti-mouse antibody for beta-actin and Bcl-2 (1:3,000; Vector Laboratories, Burlingame, CA, USA), and horseradish peroxidase-conjugated anti-rabbit antibody for Bax (1:3,000; Vector Laboratories) were used as secondary antibodies. Experiment was performed in normal lab conditions and at room temperature except membrane transfer. Membrane was transferred at 4°C with the cold pack and prechilled buffer. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection.