Recipes for buffers referenced here are as follows:
Discard the medium in the flask and wash once with pre-cold PBS.
Add 3 ml pre-cold PBS per flask and collect cells with cell scraper.
Add 12 ml pre-cold PBS to make sure all the cells detach from the flask.
Transfer collected cells to 50 ml centrifuge tubes, centrifuge at 300 x g for 5 min.
Discard supernatant, wash twice with pre-cold PBS.
Heat 1% SDS hot lysis until bubbling.
Add 1% SDS hot cell lysis according to the cell amount to resuspend cells.
Use ultrasonic cell disruptor to break all cell clusters until the lysate becomes clear.
Centrifuge for 5-10 min at 15000-17000 x g and discard cell pellet.
Discard the medium in the flask and wash once with pre-cold PBS.
Add 3 ml pre-cold PBS per flask and collect cells with cell scraper.
Add 12 ml pre-cold PBS to make sure all the cells detach from the flask.
Transfer collected cells to 50 ml centrifuge tubes, centrifuge at 300 x g for 5-10 minutes.
Discard supernatant, wash twice with pre-cold PBS.
Add RIPA buffer according to the cell amount to resuspend cells.
Use ultrasonic cell disruptor to break all cell clusters until the lysate becomes clear.
Centrifuge for 5-10 min at 15000-17000 x g and discard cell pellet.