1. Adherent and Suspension Cell Culture
- Use three T25 flasks or one T75 flask for cell culture, the number of cells (1x107);
- Suspension cell: centrifuge at 2500 rpm at 2-8℃ for 5 minutes; collect clarified cell culture supernatant;
- Adherent cell: collect supernatant directly; centrifuge at 2500 rpm at 2-8℃ for 5 minutes; collect clarified cell culture supernatant for immediate detection or store it separately at -80℃.
2. Cell Lysate Preparation
Two types of cell lysates are specified below.
2.1. Suspension Cell Lysate
Centrifuge at 2500 rpm at 2-8℃ for 5 minutes; Then add pre-cooling PBS into collected cell and gently mix. Recollect cell by repeating centrifugation. Add 0.5-1ml RIPA lysis buffer (NP-40 lysis buffer or Triton X-100 surfactant is not recommended due to the interfering with antigen-antibody reaction). Add suitable protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on the ice for 30min-1h. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Alternatively, cells are subject to fragmentation by ultrasonic cell disruptor (300W, 3~5 s/time, 30s intervals, four-five times) or ultrasonic generator (14μm for 30s ) . At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube and stored at -80℃.
2.2. Adherent Cell Lysate
Absorb supernatant and add pre-cooling PBS once. Then, add 0.5-1ml RIPA lysis buffer (NP-40 lysis buffer or Triton X-100 surfactant is not recommended due to the interfering with antigen-antibody reaction). Add the suitable protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape adherent cell gently with a cell scraper. Add the cell suspension into centrifugal tube. Lyse the cell on the ice for 30min-1h. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Alternatively, cells are subject to fragmentation by ultrasonic generator (14μm for 30s ) or ultrasonic cell disruptor (300W, 3~5 s/time, 30s intervals, four-five times). At the end of lysate/ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube and stored at -80℃.
REFERENCES
[1] Preparation of Cell Lysates of Fission Yeast for Immunoprecipitation, PMID: 29423853.
[2] Comparison of different methods of cell lysis and protein measurements in Clostridium perfringens: application to the cell volume determination, PMID: 9516540.