Alliance mini hd9 chemiluminescence documentation system

Cellular proteins were prepared from the cell pellets using lysis buffer (iNtRON Biotechnology, Seongnam, Korea). To obtain nuclear and cytoplasmic fractions, a NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA) was used according to the manufacturer’s instructions. The protein amount was measured using BCA protein assay kits (Pierce, Rockford, IL, USA). Lysates were separated on 10% SDS–polyacrylamide gel and electrophoretically transferred to PVDF membrane (Millipore Corporation, Bedford, MA), followed by blocking with 5% non-fat dry milk in PBS-Tween-20 (0.1%, vol/vol) for an hour. The membrane was incubated with a primary antibody at 4 °C for overnight. After being washed with TBS containing 0.1% Tween-20, the membrane was incubated for 1 h at room temperature with a secondary antibody. The target proteins were detected using the chemiluminescent reagents and visualized in Alliance-Mini.HD9 chemiluminescence documentation system (UVItec Cambridge, UK).

Cellular proteins were extracted using lysis buffer (iNtRON Biotechnology, Seongnam, Korea) and proteins were quantitated using BCA protein assay kits (Pierce, Rockford, IL, USA). Cell lysates were run on a 10% SDS-polyacrylamide gel, and proteins were transferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA) and blocked with 5% non-fat dry milk in PBS-Tween-20 (0.1%, v/v) for 1 h. The membranes were then probed with specific primary antibodies. After overnight incubation at 4 °C and washing with TBS containing 0.1% Tween-20, the membranes were incubated for 1 h with corresponding secondary antibodies. The target proteins were visualized with an Alliance-Mini.HD9 chemiluminescence documentation system (UVItec Cambridge, UK).