1.1.1 Conventional Mammalian Adherent Cells

Operational Steps for Protein Extraction from Conventional Mammalian Adherent Cells

1. Discard Medium/Rinse
Gently pour off the medium in the petri dish or culture flask, and aspirate the residual liquid. Add 1 mL of pre-cooled PBS to a 10 cm petri dish, rinse the cells 3 times to remove residual medium, then aspirate the PBS completely.
2. Lysis
Place the petri dish on ice. Add 500 μL of cell lysis buffer to a 10 cm petri dish (note: protease and phosphatase inhibitors must be added to the lysis buffer in advance). Incubate on ice for 10–20 minutes for lysis.
3. Sample Collection
After lysis, quickly scrape off the cells with a clean cell scraper. Collect the cell debris and lysis solution, and transfer them to a 1.5 mL EP tube. Perform the entire process on ice.
4. Centrifuge to Collect Supernatant
Centrifuge at 12,000 rpm at 4°C for 5 minutes. Collect the supernatant, aliquot it, and store at -20°C or -80°C for later use.