Horseradish peroxidase hrp conjugated secondary antibodies anti rabbit

The protein expression of SIRT2, bcl-2, and bax was assayed by western blotting. Briefly, the cerebral cortex, cerebellum, and hippocampus tissue lysates were prepared on ice with 300 μl of RIPA Lysis Buffer per 20 mg of tissue (Santa Cruz Biotechnology, TX). The samples containing 20 µg of total protein were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% or 10%). The protein bands were then transferred to nitrocellulose membranes (Bio-Rad, CA) blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% (w/v) non-fat dry milk (Santa Cruz Biotechnology, TX) for overnight at +4 °C. Following the transfer of the proteins, the membranes were incubated with primary antibodies against SIRT2 (1:500, sc-28298; Santa Cruz Biotechnology, TX), bcl-2 (1:1000, MA1-12246), bax (1:500, sc-493), or β-actin (1:125, sc-130657; Santa Cruz Biotechnology, TX) for 2 hours. Then the membranes were incubated for 1 hour with horseradish peroxidase (HRP) conjugated secondary antibodies (anti-rabbit, 1:5000, sc-2004; or anti-mouse, 1:5000, sc-2005; Santa Cruz Biotechnology, TX) in room temperature. Enhanced chemiluminescence detection reagent (Pierce™ Thermo Sci, IL) was used to visualize the specific protein bands on X-ray film (Carestream Health Inc. NY). The bands were quantified by using ImageJ software.