How do I prepare samples for the western blot experiment?

Prepare the lysis buffer, start by adding protease and phosphatase inhibitors to disrupt the cell membrane and solubilize the proteins. The choice of lysis buffer may depend on the specific proteins of the experiment.

For adherent cells:

1. Place the cell culture dish on ice and carefully remove the culture medium. Wash the cells with ice-cold PBS
2. Aspirate the PBS and add ice-cold lysis buffer (approximately 1 mL per 10^7 cells or per 100 mm plate; about 0.5 mL for a 60 mm plate; approximately 200-400 µL for a 6-well culture plate). Swirl the dish for 5 minutes on ice
3. Gather the lysate and transfer it to a microcentrifuge tube. Centrifuge the samples at approximately 14,000 x g for 15 minutes to pellet the cell debris
4. Transfer the supernatant to a new microcentrifuge tube and throw away the pellet

For suspension-cultured cells:

1. Pellet the suspension of cells through centrifugation at 2,500 x g for 10 minutes. Carefully discard the supernatant, leaving behind the cell pellet.
2. Wash the cells once by resuspending the cell pellet in ice-cold phosphate-buffered saline (PBS). Centrifuge the suspension again at 2,500 x g for 10 minutes to pellet the cells.
3. Add ice-cold lysis buffer to the cell pellet (approximately 1 mL per 100 mg of pellet or  100 µL of wet cell pellet). Ensure thorough resuspension of the pellet by pipetting the mixture up and down.
4. Gently shake the mixture for 10 minutes to facilitate lysis of the cells. Centrifuge the lysate at 14,000 x g for 15 minutes to pellet any remaining cell debris.
5. Carefully transfer the supernatant containing the protein lysate to a new microcentrifuge tube and throw away the pellet.