4.12. Western Blot

Mouse-monoclonal antibody β and Actin antibody (1:5000) were used as primary antibodies and revealed at 18 s of exposure. Mouse-monoclonal antibody p21 (1:100) was also used and revealed at 34 s of exposure. Antibodies were acquired from Santa Cruz Biotechnology (sc-47778 and sc-6246, respectively). The procedure was performed following the previously reported protocol [34]. Protein was extracted from the tumors, for which the tumors were crushed with an extraction buffer: 200 μL protease inhibitor, 800 μL lysis buffer, and PMSF 10 μL phosphatase inhibitor. After homogenization, they were centrifuged for 30 min at 4 °C, RFC 8000. The protein content was evaluated in the supernatant. The total protein content was determined using the Thermo Scientific Pierce BCA Protein Assay Kit (catalog number 23225) in 96-well plates at 570 nm. The samples were heated at 95 °C for 15 min in a thermocycler for protein denaturation. A total of 40 μg of protein was separated on an SDS page. Afterwards, the gels were transferred to polyvinylidene difluoride (PVDF) membranes (0.2 μM pore size). After the electrotransfer, the membranes were blocked with 5% BSA in TBST for 1 h. Subsequently, the primary antibody (mouse-monoclonal antibody p21) in 5% bovine serum albumin (BSA) was added and incubated overnight at 4 °C on a shaker. The next day the membrane was washed with TBST for 5 min for 3 times under stirring, and the secondary antibody was added and incubated for 1 h. The membrane was then washed with TBST for 5 min for 2 times and once again with TBS without Tween. Signal was development in the dark room.