Molecular image chemidoc xrs system

The cells in the high-speed growth phase were treated with a 4-fold dilution of the Mg alloy extract and medium for 24 h, washed with PBS three times and added RIPA cell lysate containing 1% protease inhibitor and 1% phosphatase inhibitor. A cell scraper was used to hang the adherent cells. The lysate was collected, vortexed and placed on ice for 30 min to be fully lysed. After 12 000 g refrigerated centrifugation for 10 min, the supernatant was transferred to a clean EP tube. The Micro BCA™ Protein Assay Kit (Thermo Scientific, USA) was used to measure the protein concentration of the samples, and then a 5× loading buffer was added. The samples were heated at 100°C for 15 min to denature the proteins. The proteins were separated by using 10% SDS-PAGE, and the same amount of total protein (20 μg) was added to the loading well. After the electrophoresis, the proteins were transferred to the PVDF membrane, which was blocked with 5% skim milk for 2 h, incubated with the primary antibody (Table 2) at 4°C overnight, then washed 3 times for 5 min with TBST buffer. The secondary antibody was used to incubate for 2 h at room temperature. After washing with TBST three times, the targeted proteins were visualized with enhanced chemiluminescence (ECL, Beyotime Biotechnology Co., China) in the Molecular Image ChemiDoc XRS⁺ system (Bio-Rad Laboratories Inc., USA).