Quantifying Kidney Protein Profiles

Proteins were extracted from kidney, and the protein concentrations were measured using a BCA assay kit (TaKaRa BIO INC, Japan). The protein samples were resolved in a 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membrane, and blocked with 5% nonfat milk in Tris-buffered saline-Tween (TBST) 20 for 2 h at room temperature. Membranes were then incubated with primary antibody overnight. The antibodies were shown as follows: anti-Caspase-3 (1:1000; Santa Cruz Biotechnology), or anti-HSP70 (1:1000; Santa Cruz Biotechnology). An anti-β-actin antibody was used as control. Membranes were washed and incubated for 2 h in the presence of appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The positive reaction was visualized by using 3, 3′-diaminobenzitine (DAB) solution (Sigma, St. Louis, MO) with a chemiluminescent Immobilon Western blotting detection system^{16 (link),17 (link)}.