Mini protean tgx stain free precast protein gel

hWJ-MSCs were lysed using Mammalian Protein Extraction Reagent (M-PER, Thermo Fisher Scientific, San Diego, CA, USA) containing protease and phosphate inhibitors (Sigma-Aldrich Co., St. Louis, MO, USA). Protein concentrations were measured using Bradford Reagent (Sigma-Aldrich Co., St. Louis, MO, USA). Cellular lysates were resuspended in 4× Laemmli Sample Buffer and incubated at 95 °C for 5 min. Twenty micrograms of cellular lysates were separated to SDS-PAGE on a 10% Mini-PROTEAN^® TGX Stain-Free™ Precast Protein Gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to a 0.2 µm nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking, nitrocellulose membrane was incubated with caspase 3 primary antibody (1:1000 dilution; sc-7148 Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4 °C, and then probed with horseradish peroxidase (HRP) conjugated secondary antibody (1:10,000 dilution; AbCam, Cambridge, UK) for 1 h at RT. Bound antibodies were detected with the use of Clarity Western ECL Substrate. The sample loading control was evaluated by a stain-free detection of proteins in the gel after electrophoresis.