Bca analysis kit

Chondrocytes were inoculated into six-well plates at a density of 5 × 10⁵ cells per well and allowed to adhere for 48 hours. First, the cells were treated in different groups for 24 hours. The cells were washed twice with phosphate-buffered saline (PBS), and then RIPA lysis buffer containing a 1% protease inhibitor mixture (AR0102; Boster, China) was used to lyse chondrocytes for 30 minutes on ice. The extract was collected and subjected to centrifugation at a speed of 12,000× g and a temperature of 4°C for 30 minutes. Then, a BCA analysis kit (AR0146; Boster) was used to determine the protein concentration of cell lysates. Next, the supernatant of each sample was collected, and then the samples (25 μg) were separated through electrophoresis on a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (MilliporeSigma, USA). After being blocked with 5% skimmed milk at ordinary temperature for one hour, the membranes and specific primary antibodies were incubated at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies at room temperature for one hour. To visualize the protein bands, ultra-sensitive ECL chemiluminescence reagent from Boster was employed. The resulting images were captured using the ChemiDocTM XRS+ System (Bio-Rad Laboratories, USA).