Mini protein tgx precast gel

Whole cell lysates were collected using urea lysis buffer containing 6.6M urea, 10mM Tris-HCl, 1% SDS, 5mM DTT, 1% Triton X-100, 10% Glycerol and 1 × protein inhibitor mix. Protein concentrations were determined by using Bio-Rad Protein Assay Kit I. The same amounts of total proteins were loaded onto a Mini-protein TGX precast gel (Bio-Rad, Hercules, CA) and electrophoresis was performed. The separated proteins were transferred onto PVDF membrane (Bio-Rad), blocked with 5% non-fat dry milk powder in TBST buffer (50mM Tris, pH 8.0, 150mM NaCl, 0.2% Tween-20), and probed with the appropriate antibody. Bands were visualized using chemiluminescence system (Thermo Scientific, Rockford, IL). Western blots were stripped in buffer containing 62.5mMTris-HCl, pH 6.7, 2% SDS, and 100mM 2-mercaptoethanol at 50°C for 1 h, washed 4–6 times in TBST buffer, and re-probed when needed. Western blot quantification was performed using Quantity One (Bio-Rad); the bands were automatically analyzed by the software. Signals were calculated by subtracting the detected intensity × mm² by background. Signal from control was arbitrarily set as 1.00, while the signals of treated groups were denoted as the fold compared to the control.