Lysate for Western-PDF

Protocol

1. Grow cells
2. Harvest
   • Spin down
   • Wash with PBS
   • Spin down enough cells to collect a 50-100 µL cell pellet in a FastPrep tube
3. Prepare lysis buffer (beforehand) and chill on ice
   • PBSMT:
     • 2.5 mM MgCl₂
     • 3 mM KCl
     • 0.5% Triton X-100
     • in PBS
   • Add protease inhibitors
     • PMSF: 1 mL per 100 mL buffer
       • Use fresh PMSF: dissolve 0.035g per 1mL 100%EtOH
     • PLAAC: 100 µL per 100 mL buffer
     • 0.5 M Benzamidine: 260 µL per 100 mL buffer
4. Add 50-100 µL of cold lysis buffer to cell pellet and vortex
5. Add 1-2 scoops glass beads (bead level should be just below liquid level)
6. Mix everything together by vortex
7. Lyse in FastPrep bead beater
   • 6.5 m/s
   • 40 second lysis
8. Add 500 µL lysis buffer (keep everything on ice!)
9. Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room)
10. Remove supernatant (lysate) to another tube.