Example Protocol for Cell Lysing :

  1. Remove the culture media by aspiration.
  2. Wash cells with cold PBS and aspirate off PBS (1X). Add cold NP40 Cell Lysis Buffer (RIPA Lysis Buffer: 25mM Tris-HCl pH7.5, 150mM NaCl, 1% NP-40, 1mM EDTA pH8.0. Add fresh: 1 mM PMSF, 1 mM Na3VO4, and 1 X Protease Inhibitor Cocktail-P2714, Sigma).
  3. When cells are over 70% confluentwe recommend adding 0.8 to 1 ml NP40 Cell Lysis buffer for 10-cm cell culture dish.
  4. For cells that are adherent , scrap cells using cell scraper under ice. For suspension cells, pellet the cells, then resuspend in lysis buffer under ice.
  5. Transfer the cell lysis solution into eppendorftype tubes after pipetting the cell pellets up to15 times.
  6. Sonicate the cell lysates under ice for 10 sec.
  7. Centrifuge the lysate at 12,000g in a pre-cooled centrifuge for 15 minutes.
  8. When centrifugation is completetransfer the supernatant to a fresh centrifuge tube and discard the pellet.
  9. Calculatethe protein concentration
  10. The sample can now be divided into aliquots  and stored at –80o C for long-term.