Any kd tris glycine gels

The precipitated proteins were mixed with β-mercaptoethanol, incubated in a boiling water bath for five minutes, and loaded onto precast Any-kD Tris-glycine gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) for electrophoresis so that each well contained 60 μg of creatinine per sample. Normalization to creatinine with urine stored in these conditions is the standard for urinary protein processing via western blotting [16 (link)]. Proteins were transferred onto PVDF membranes and blocked with 5% nonfat milk in PBS-Tween 20. Blocked membranes were incubated overnight with anti-AQP1 (H-55) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), diluted 1 : 500 in blocking buffer. After washing, the membranes were incubated with a 1 : 2000 dilution of goat anti-rabbit IgG HRP-conjugated antibody (Cell Signaling Technology, Danvers, MA, USA) for two hours at room temperature and visualized by chemiluminescence. AQP1 levels were semiquantified in arbitrary units using ImageJ software for area under the curve (AUC) analysis.